Singh, Geetika; Singh, Lavleen; Ghosh, Ranajoy; Nath, Devajit; Dinda, Amit Kumar
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
Wagrowska-Danilewicz, Małgorzata; Danilewicz, Marian
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.
Tasdemir, S; Eroz, R; Cucer, N; Oktay, M; Türkeli, M
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.
Nada, R; Kumar, A; Kumar, V G; Gupta, K L; Joshi, K
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.
Mubarak, Muhammed; Kazi Javed, I; Kulsoom, Umme; Ishaque, Muhammed
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
Tfayli, Ali; Gobinet, Cyril; Vrabie, Valeriu; Huez, Regis; Manfait, Michel; Piot, Olivier
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).
Sergio, Navarro-Fierros; Cecilia, Guillen-Vargas; Sonia, Luquin; la Mora Pedro, Garzon-De; Joaquin, Garcia-Estrada; Mary, Fafutis-Morris
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.
Morbini, Patrizia; Alberizzi, Paola; Tinelli, Carmine; Paglino, Chiara; Bertino, Giulia; Comoli, Patrizia; Pedrazzoli, Paolo; Benazzo, Marco
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.
Tóth-Lipták, Judit; Piukovics, Klára; Borbényi, Zita; Demeter, Judit; Bagdi, Enikő; Krenács, László
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.
Chaubert, Pascal; Baur Chaubert, Audrey S; Sattler, Ursula; Forster, Ursula; Bornand, Valérie; Suter, Maja; Welle, Monika
In the dog, early-stage epitheliotropic T-cell lymphoma (ETCL) can clinically and histologically mimic a large range of inflammatory dermatoses and often progresses rapidly to a more aggressive tumor stage. Early diagnosis of ETCL is essential to proceed with a specific oncologic therapy that is favorable for the prognosis. In the present study, an improved method for the detection of T-cell receptor gamma (TCRgamma) rearrangement was developed by designing a new set of consensus primers to amplify the different forms of rearranged canine TCRgamma gene sequences by polymerase chain reaction. The amplicons were analyzed by conventional polyacrylamide gel electrophoresis, which requires minimal specific equipment and may be performed in almost every pathology laboratory at low costs. The method proved to be highly specific and sensitive to detect early ETCL in formalin-fixed, paraffin-embedded biopsy specimens, providing an efficient tool for veterinary pathologists to distinguish early neoplastic from reactive cutaneous T-cell infiltrates (tumor-specific marker) or to discriminate T-cell lymphoma from B-cell lymphomas or nonlymphoid neoplasms (T-cell lineage marker). By direct sequencing analysis of amplified TCRgamma gene sequences, ETCL was found to rearrange exclusively the joining (J) 4 region, which suggests specific biology for primary cutaneous T-cell lymphomas. Also, a novel (seventh) functional J region in the TCRgamma gene, localized approximately 2.3 kb upstream of J5, was identified.
Charef, S; Jrad, B Bel Hadj; Mahfouth, W; Zakhama, A; Kassab, A; Driss, N; Chouchane, L
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.
Monaghan, Michael G.; Kroll, Sebastian; Brucker, Sara Y.
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
Lanús, Elizabeth Córdoba; Piñero, José Enrique; González, Ana Cristina; Valladares, Basilio; de Grosso, Mercedes Lizarralde; Salomón, Oscar Daniel
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.
Chung, Joon-Yong; Cho, Hanbyoul; Hewitt, Stephen M
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.
Stockert, J C; López-Arias, B; Del Castillo, P; Romero, A; Blázquez-Castro, A
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.
Casadonte, Rita; Caprioli, Richard M
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
Brinkmann, Volker; Abu Abed, Ulrike; Goosmann, Christian; Zychlinsky, Arturo
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
Valbuena, Carmen; Leitão, Dina; Carneiro, Fátima; Oliveira, João Paulo
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.
Araújo-Pérez, Félix; McCoy, Amber N; Okechukwu, Charles; Carroll, Ian M; Smith, Kevin M; Jeremiah, Kim; Sandler, Robert S; Asher, Gary N; Keku, Temitope O
There is growing evidence the microbiota of the large bowel may influence the risk of developing colorectal cancer as well as other diseases including type-1 diabetes, inflammatory bowel diseases and irritable bowel syndrome. Current sampling methods to obtain microbial specimens, such as feces and mucosal biopsies, are inconvenient and unappealing to patients. Obtaining samples through rectal swabs could prove to be a quicker and relatively easier method, but it is unclear if swabs are an adequate substitute. We compared bacterial diversity and composition from rectal swabs and rectal mucosal biopsies in order to examine the viability of rectal swabs as an alternative to biopsies. Paired rectal swabs and mucosal biopsy samples were collected in un-prepped participants (n = 11) and microbial diversity was characterized by Terminal Restriction Fragment Length polymorphism (T-RFLP) analysis and quantitative polymerase chain reaction (qPCR) of the 16S rRNA gene. Microbial community composition from swab samples was different from rectal mucosal biopsies (p = 0.001). Overall the bacterial diversity was higher in swab samples than in biopsies as assessed by diversity indexes such as: richness (p = 0.01), evenness (p = 0.06) and Shannon's diversity (p = 0.04). Analysis of specific bacterial groups by qPCR showed higher copy number of Lactobacillus (p < 0.0001) and Eubacteria (p = 0.0003) in swab samples compared with biopsies. Our findings suggest that rectal swabs and rectal mucosal samples provide different views of the microbiota in the large intestine.
Koonmee, Supinda; Intapan, Pewpan M; Yamasaki, Hiroshi; Sugiyama, Hiromu; Muto, Maki; Kuramochi, Toshiaki; Kularbkeaw, Jurairat; Kanpittaya, Jaturat; Maleewong, Wanchai; Nawa, Yukifumi
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.
Bonin, Serena; Stanta, Giorgio
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.
Signoretti, Sabina; Murphy, Michael; Cangi, Maria Giulia; Puddu, Pietro; Kadin, Marshall E.; Loda, Massimo
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
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.
Godon, Alban; Genevieve, Franck; Valo, Isabelle; Josselin, Nicolas; Talmant, Pascaline; Foussard, Charles; Avet-Loiseau, Herve; Ifrah, Nobert; Zandecki, Marc; Rousselet, Marie-Christine
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.
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...
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...
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...
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...
Choi, Yun Sik; Kim, Yong Cheol; Baek, Keum Jin; Choi, Youngnim
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.
Frickmann, Hagen; Tenner-Racz, Klara; Eggert, Petra; Schwarz, Norbert G; Poppert, Sven; Tannich, Egbert; Hagen, Ralf M
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.
Tanzi, Elisabetta; Bianchi, Silvia; Frati, Elena R; Amicizia, Daniela; Martinelli, Marianna; Bragazzi, Nicola L; Brisigotti, Maria Pia; Colzani, Daniela; Fasoli, Ester; Zehender, Gianguglielmo; Panatto, Donatella; Gasparini, Roberto
Human papillomavirus (HPV) has a well-recognized aetiological role in the development of cervical cancer and other anogenital tumours. Recently, an association between colorectal cancer and HPV infection has been suggested, although this is still controversial. This study aimed at detecting and characterizing HPV infection in 57 paired biopsies from colorectal cancers and adjacent intact tissues using a degenerate PCR approach. All amplified fragments were genotyped by means of sequencing. Overall, HPV prevalence was 12.3 %. In particular, 15.8 % of tumour tissues and 8.8 % of non-cancerous tissue samples were HPV DNA-positive. Of these samples, 85.7 % were genotyped successfully, with 41.7 % of sequences identifying four genotypes of the HR (high oncogenic risk) clade Group 1; the remaining 58.3 % of HPV-genotyped specimens had an unclassified β-HPV. Examining additional cases and analysing whole genomes will help to outline the significance of these findings.
Noda, Angel Alberto; Rodríguez, Islay; Rodríguez, Yaindrys; Govín, Anamays; Fernández, Carmen; Obregón, Ana Margarita
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
Long, Delwin J; Buggs, Colleen
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.
Kaneko, Y.; Onda, N.; Watanabe, Y.; Shibutani, M.
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
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
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.
Spolidorio, Luís Carlos; Martins, Vinícius Rangel; Nogueira, Ruchele Dias; Spolidorio, Denise Madalena
Candidosis is the most common fungal infection in the oral cavity, and is usually associated with local and systemic predisposing factors. The occurrence and relevance of Candidal infection in oral lesions such as liquen planus, leukoplakias and carcinomas are still to be understood. The aim of the present study was to define the frequency of infection by Candida sp. on biopsies of oral mucosal lesions and associate its presence with malignant and dysplastic lesions. Histopathology reports issued between 1990 and 2001 inclusive were reviewed. Three sections of each mucosal biopsy were stained using the periodic acid-Schiff (PAS) technique. From the 832 biopsies 27.2% were PAS positive, of which 83.25% were obtained from male patients. There was positive association between fungic infection and mild, moderate and severe epithelial dysplasia, squamous cell carcinoma and hiperqueratosis (p < 0.05). There was no association between fungic infection and inflammatory fibrous hyperplasia, hyperkeratosis, lichen planus and pyogenic granuloma (p < 0.05). The frequency of infection in the tongue was significantly higher (p < 0.05) than in the other sites. Our results do not show a causal relation between Candida sp. and dysplastic lesions and carcinomas, but do confirm the higher presence of that microrganism in those lesions.
Renwick, Neil; Cekan, Pavol; Bognanni, Claudia; Tuschl, Thomas
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.
Navarro, J A; Ortega, N; Buendia, A J; Gallego, M C; Martínez, C M; Caro, M R; Sánchez, J; Salinas, J
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.
Renwick, Neil; Cekan, Pavol; Bognanni, Claudia; Tuschl, Thomas
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
Vogel, Benjamin; Siebert, Hanna; Hofmann, Ulrich; Frantz, Stefan
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
Ilgen, Peter; Stoldt, Stefan; Conradi, Lena-Christin; Wurm, Christian Andreas; Rüschoff, Josef; Ghadimi, B Michael; Liersch, Torsten; Jakobs, Stefan
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.
Kelley, Andrea R.; Perry, George; Bethea, Chloe; Castellani, Rudolph J.; Bach, Stephan B.H.
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
Zeng, Hanxiang; Li, Xiqing; Chen, Xiejie; Zhang, Junmin; Sun, Jiufeng; Xie, Zhi; Xi, Liyan
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.
Mesquita, R A; Anzai, E K; Oliveira, R N; Nunes, F D
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.
Donà, Maria Gabriella; Ronchetti, Livia; Giuliani, Massimo; Carosi, Mariantonia; Rollo, Francesca; Congiu, Mario; Mazza, Domenica; Pescarmona, Edoardo; Vocaturo, Amina; Benevolo, Maria
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.
Stiller, Mathias; Sucker, Antje; Griewank, Klaus; Aust, Daniela; Baretton, Gustavo Bruno; Schadendorf, Dirk; Horn, Susanne
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
Mahmoodi, Pezhman; Motamedi, Hossein; Seyfi Abad Shapouri, Masoud Reza; Bahrami Shehni, Mahjabin; Kargar, Mohammad
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
Lück, P C; Helbig, J H; Witzleb, W
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.
Chiu, Wai Kan; Brand, Rhonda M; Camp, Danielle; Edick, Stacey; Mitchell, Carol; Karas, Sherri; Zehmisch, Amanda; Ho, Ken; Brand, Randall E; Harrison, Janet; Abo, Steven; Cranston, Ross D; McGowan, Ian
During Phase 1 pharmacokinetic/pharmacodynamics studies, participants may undergo multiple sigmoidoscopies, with a collection of 10-20 biopsies during each procedure. This article characterizes the safety of flexible sigmoidoscopies in clinical trial participants. We determined the number of flexible sigmoidoscopies and rectal biopsies that participants underwent and analyzed the frequency, duration, and severity of flexible sigmoidoscopy-related adverse events (AEs). During the study period, 278 participants underwent 1,004 flexible sigmoidoscopies with the collection of 15,930 rectal biopsies. The average number of procedures per participant was 3.6 (median 3; range 1-25), with an average time interval between procedures of 61.8 days (median 28 days; range 1-1,159). There were no serious AEs. Sixteen AEs were related to flexible sigmoidoscopy and occurred in 16 participants, leading to an overall 1.6% (16/1,004) AE rate per procedure and 0.1% (16/15,930) AE rate per biopsy. Of the 16 AEs, 8 (50%) involved abdominal pain, diarrhea, bleeding, flatulence, and bloating, with an average duration of 4.7 days (median 1 day; range 1-28). Most (14/16) AEs were categorized as Grade 1 (mild), whereas two of the AEs were Grade 2 (moderate). No participant withdrew due to procedure-related AEs. Overall, the number of AEs caused by flexible sigmoidoscopy with multiple biopsies was low and the severity was mild, suggesting that this procedure can be safely integrated into protocols requiring repeated intestinal mucosal sampling.
Robertson, David; Isacke, Clare M
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.
Porter, Amy; Irwin, Regina; Miller, Josselyn; Horan, Daniel J.; Robling, Alexander G.; McCabe, Laura R.
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
Wahaia, Faustino; Kasalynas, Irmantas; Seliuta, Dalius; Molis, Gediminas; Urbanowicz, Andrzej; Carvalho Silva, Catia D.; Carneiro, Fatima; Valusis, Gintaras; Granja, Pedro L.
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.
Rivero, Elena R C; Neves, Adriana C; Silva-Valenzuela, Maria G; Sousa, Suzana O M; Nunes, Fabio D
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.
Reshef, Ayeleth; Barash, Mark; Voskoboinik, Lev; Brauner, Paul; Gafny, Roni
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.
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
Wahaia, Faustino; Kasalynas, Irmantas; Seliuta, Dalius; Molis, Gediminas; Urbanowicz, Andrzej; Carvalho Silva, Catia D.; Carneiro, Fatima; Valusis, Gintaras; Granja, Pedro L.
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.
Ballester, Maria; Rodríguez, Fernando
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.
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...
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...
Oberauner-Wappis, Lisa; Loibner, Martina; Viertler, Christian; Groelz, Daniel; Wyrich, Ralf; Zatloukal, Kurt
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.
Seldenrijk, C A; Morson, B C; Meuwissen, S G; Schipper, N W; Lindeman, J; Meijer, C J
In a prospective blind evaluation of multiple colonic mucosal biopsy specimens, 45 clinically well defined patients with chronic inflammatory bowel disease (21 Crohn's disease and 24 ulcerative colitis) and 16 control subjects (seven normal subjects and nine patients with diverticular disease) were studied to identify reproducible histopathological features which could distinguish chronic inflammatory bowel disease (CIBD) from non-CIBD and Crohn's disease from ulcerative colitis. Using kappa statistics 16 of 41 histological features were sufficiently reproducible for further stepwise discriminant analysis to differentiate between CIBD and non-CIBD, and between Crohn's disease and ulcerative colitis. Using the combination of three features (an increase of lymphocytes and plasma cells in the lamina propria, the presence of branching of crypts, and neutrophils in the crypt epithelium) we were able to distinguish CIBD from non-CIBD in 89% of the cases with high probability (p greater than 0.85). To separate Crohn's disease from ulcerative colitis three features (an excess of histiocytes in combination with a villous or irregular aspect of the mucosal surface and granulomas) had a high predictive value. Using these features 70% of Crohn's disease patients and 75% of ulcerative colitis patients were correctly classified with a high probability (p greater than 0.85). These findings indicate that the pathologist is dependent on the presence of only a few histological features for a reliable classification of Crohn's disease and ulcerative colitis. Images Figure 1 PMID:1773958
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
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
Chen, Shun; Cheng, Anchun; Wang, Mingshu; Zhu, Dekang; Luo, Qihui; Liu, Fei; Chen, Xiaoyue
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
... News Physician Resources Professions Site Index A-Z Biopsies - Overview A biopsy is the removal of tissue ... What are the limitations of biopsies? What are biopsies? A biopsy is the removal of tissue in ...
Adam, Benjamin; Afzali, Bahman; Dominy, Katherine M; Chapman, Erin; Gill, Reeda; Hidalgo, Luis G; Roufosse, Candice; Sis, Banu; Mengel, Michael
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.
Barranco, Inmaculada; Gómez-Laguna, Jaime; Rodríguez-Gómez, Irene M; Salguero, Francisco J; Pallarés, Francisco J; Bernabé, Antonio; Carrasco, Librado
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.
Tanca, Alessandro; Addis, Maria Filippa; Pagnozzi, Daniela; Cossu-Rocca, Paolo; Tonelli, Roberto; Falchi, Giovanni; Eccher, Albino; Roggio, Tonina; Fanciulli, Giuseppe; Uzzau, Sergio
Hospital tissue repositories host an invaluable supply of diseased samples with matched retrospective clinical information. In this work, a recently optimized method for extracting full-length proteins from formalin-fixed, paraffin-embedded (FFPE) tissues was evaluated on lung neuroendocrine tumor (LNET) samples collected from hospital repositories. LNETs comprise a heterogeneous spectrum of diseases, for which subtype-specific diagnostic markers are lacking. Six archival samples diagnosed as typical carcinoid (TC) or small cell lung carcinoma (SCLC) were subjected to a full-length protein extraction followed by a GeLC-MS/MS analysis, enabling the identification of over 300 distinct proteins per tumor subtype. All identified proteins were categorized through DAVID software, revealing a differential distribution of functional classes, such as those involved in RNA processing, response to oxidative stress and ion homeostasis. Moreover, using spectral counting for protein abundance estimation and beta-binomial test as statistical filter, a list of 28 differentially expressed proteins was generated and submitted to pathway analysis by means of Ingenuity Pathway Analysis software. Differential expression of chromogranin-A (more expressed in TCs) and stathmin (more expressed in SCLCs) was consistently confirmed by immunohistochemistry. Therefore, FFPE hospital archival samples can be successfully subjected to proteomic investigations aimed to biomarker discovery following a GeLC-MS/MS label-free approach.
Creech, Matthew K.; Wang, Jing; Nan, Xiaolin; Gibbs, Summer L.
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
Wang, Jian-Hua; Gouda-Vossos, Amany; Dzamko, Nicolas; Halliday, Glenda; Huang, Yue
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.
Potluri, Keerti; Mahas, Ahmed; Kent, Michael N; Naik, Sameep; Markey, Michael
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.
Kocjan, Boštjan J; Hošnjak, Lea; Poljak, Mario
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.
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
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
Menon, Roopika; Deng, Mario; Boehm, Diana; Braun, Martin; Fend, Falko; Boehm, Detlef; Biskup, Saskia; Perner, Sven
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.
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
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.
Creech, Matthew K; Wang, Jing; Nan, Xiaolin; Gibbs, Summer L
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.
Drummond, Eleanor S; Nayak, Shruti; Ueberheide, Beatrix; Wisniewski, Thomas
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.
Faílde, L D; Bermúdez, R; Losada, A P; Riaza, A; Santos, Y; Quiroga, M I
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.
Shi, Shan-Rong; Taylor, Clive R; Fowler, Carol B; Mason, Jeffrey T
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.
Shi, Shan-Rong; Liu, Cheng; Balgley, Brian M; Lee, Cheng; Taylor, Clive R
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.
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
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.
Perosio, P. M.; Brooks, J. J.
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
Menon, Roopika; Deng, Mario; Boehm, Diana; Braun, Martin; Fend, Falko; Boehm, Detlef; Biskup, Saskia; Perner, Sven
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
Wojakowska, Anna; Marczak, Łukasz; Jelonek, Karol; Polanski, Krzysztof; Widlak, Piotr; Pietrowska, Monika
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.
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
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
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
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
Belder, Nevin; Coşkun, Öznur; Erdoğan, Beyza Doğanay; Savaş, Berna; Ensari, Arzu; Özdağ, Hilal
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) ; Scicchitano et al., Scicchitano et al. (2006) ; Frank et al., Frank et al. (2007) ; Fedorowicz et al., Fedorowicz et al. (2009) ). 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
Rodriguez, Rubens; da Silva, Ana Cristina Aramburú; Müller, Carla Aristonara; Alves, Silvana Lunardini; Graeff-Teixeira, Carlos; Fornari, Fernando
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
Belder, Nevin; Coşkun, Öznur; Erdoğan, Beyza Doğanay; Savaş, Berna; Ensari, Arzu; Özdağ, Hilal
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) ; Scicchitano et al., Scicchitano et al. (2006) ; Frank et al., Frank et al. (2007) ; Fedorowicz et al., Fedorowicz et al. (2009) ). 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.
Zhang, Binxue; Wear, Douglas J; Stojadinovic, Alexander; Izadjoo, Mina
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.
Heikal, Nahla; Nussenzveig, Roberto H; Agarwal, Archana M
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).
Yağmur, Gülhan; Albayrak, Nurhan; Daş, Taner; Yıldırım, Muzaffer; Ozgün, Ayşe; Büyük, Yalçın
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
Villabona, Lisa; Leon Rodriguez, Daniel A; Andersson, Emilia K; Seliger, Barbara; Dalianis, Tina; Masucci, Giuseppe V
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.
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
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.
Korabiowska, Monika; Fischer, Gösta; Steinacker, Anja; Stachura, Jerzy; Cordon-Cardo, Carlos; Brinck, Ulrich
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.
Abdel-Glil, Mostafa Y; Mor, Sunil K; Sharafeldin, Tamer A; Porter, Robert E; Goyal, Sagar M
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.
van Duijn, Sara; Nabuurs, Rob J A; van Duinen, Sjoerd G; Natté, Remco
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.
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
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.
Yan, Jun; Catts, Vibeke S; Chan, Anthony; McCombe, Pamela A
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.
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
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.
van Essen, Hendrik F; Ylstra, Bauke
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.
Morshed, Kamal; Polz-Dacewicz, Małgorzata; Szymański, Marcin; Smoleń, Agata
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.
Janecka, Anna; Adamczyk, Agnieszka; Gasińska, Anna
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.
Bianchi, A. B.; Navone, N. M.; Conti, C. J.
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
Lin, Jianghai; Kennedy, Stephen H.; Svarovsky, Therese; Rogers, Jeffrey; Kemnitz, Joseph W.; Xu, Anlong; Zondervan, Krina T.
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
Tasaki, Masayoshi; Obayashi, Konen; Ueda, Mitsuharu; Ando, Yukio
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.
Canteros, Cristina E; Vélez H, Alejandro; Toranzo, Adriana I; Suárez-Alvarez, Roberto; Tobón O, Ángela; Jimenez A, María del Pilar; Restrepo M, Ángela
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.
Marwitz, Sebastian; Kolarova, Julia; Reck, Martin; Reinmuth, Niels; Kugler, Christian; Schädlich, Ines; Haake, Andrea; Zabel, Peter; Vollmer, Ekkehard; Siebert, Reiner; Goldmann, Torsten; Ammerpohl, Ole
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.
Muciaccia, Barbara; Vico, Carmen; Aromatario, Mariarosaria; Fazi, Francesco; Cecchi, Rossana
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.
Musella, Valeria; Callari, Maurizio; Di Buduo, Eleonora; Scuro, Manuela; Dugo, Matteo; Miodini, Patrizia; Bianchini, Giampaolo; Paolini, Biagio; Gianni, Luca; Daidone, Maria Grazia; Cappelletti, Vera
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.
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...
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...
Hua, Xia; Zhang, Ruifeng; Yang, Hanjun; Lei, Song; Zhang, Yizhi; Ran, Yuping
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.
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...
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...
... Nemcek AA. Percutaneous biopsy. In: Mauro MA, Murphy KPJ, Thomson KR, Venbrux AC, Morgan RA, eds. Image- ... by URAC, also known as the American Accreditation HealthCare Commission (www.urac.org). URAC's accreditation program is ...
Olaofe, Olaejirinde Olaniyi; Sabageh, Donatus; Komolafe, Akinwunmi Oluwole
Introduction Although it is a well recognized premalignant lesion of the stomach, there is a dearth of information on the clinicopathologic features of gastric intestinal metaplasia in Nigerians. It is, therefore, necessary to study these features and their possible contribution to the development of gastric carcinoma in Nigerians. Methods All gastric biopsies with the histo-morphologic features of intestinal metaplasia diagnosed at the department of morbid anatomy and forensic medicine, Obafemi Awolowo university teaching hospitals complex, Ile-Ife, Nigeria between January 2006 and December 2010 were used for the study. Results A total of 165 biopsies (21.3% of all gastric biopsies within the study period) with background chronic gastritis and intestinal metaplasia were reviewed. The mean age of patients with intestinal metaplasia was 50.3 years ± 17 standard deviation (SD) while the ages of the patients ranged from 10-100 years. There were 83 males (50.3%) with a mean age of 48.1 ± 18.2 SD years and 95% confidence interval (CI) of 44.1-52.1 years. There were, however, 82 females (49.6%) with a mean age of 52.5 (± 15.8 SD) years and a 95% CI of 49.0-56.0 years. There was no significant association between the histologic type of intestinal metaplasia and the patients’ sex, age groups, severity of chronic gastritis, disease activity or degree of gastric glandular atrophy. Conclusion There are no statistically significant differences in the clinicopathologic characteristics of the subtypes of intestinal metaplasia. In majority of patients, progression from intestinal metaplasia to gastric adenocarcinoma probably takes an average of about 7 years. PMID:27217900
Sayej, Wael N; Ménoret, Antoine; Maharjan, Anu S; Fernandez, Marina; Wang, Zhu; Balarezo, Fabiola; Hyams, Jeffrey S; Sylvester, Francisco A; Vella, Anthony T
Eosinophilic esophagitis (EoE) is an emerging allergic, IgE- and non-IgE (Th2 cell)-mediated disease. There are major gaps in the understanding of the basic mechanisms that drive the persistence of EoE. We investigated whether esophageal biopsies from children with EoE demonstrate an inflammatory response that is distinct from normal controls. We prospectively enrolled 84 patients, of whom 77 were included in our analysis, aged 4–17 years (12.8±3.8 years; 81% males). Five esophageal biopsies were collected from each patient at the time of endoscopy. Intramucosal lymphocytes were isolated, phenotyped and stimulated with phorbol 12-myristate 13-acetate/ionomycin to measure their potential to produce cytokines via flow cytometry. We also performed cytokine arrays on 72-h biopsy culture supernatants. CD8+ T cells, compared with CD4+ T cells, synthesized more TNF-α and interferon (IFN)-γ after mitogen stimulation in the EoE-New/Active vs EoE-Remission group (P=0.0098; P=0.02) and controls (P=0.0008; P=0.03). Culture supernatants taken from explant esophageal tissue contained 13 analytes that distinguished EoE-New/Active from EoE-Remission and Controls. Principal component analysis and cluster analysis based on these analytes distinctly separated EoE-New/Active from EoE-Remission and Controls. In summary, we have identified a previously unappreciated role for CD8+ T lymphocytes with potential to produce TNF-α and IFN-γ in EoE. Our results suggest that CD8+ T cells have a role in the persistence or progression of EoE. We have also identified a panel of analytes produced by intact esophageal biopsies that differentiates EoE-New/Active from EoE-Remission and controls. Our results suggest that esophageal epithelial cells may have specific immune effector functions in EoE that control the type and amplitude of inflammation. PMID:27525061
Uzal, F A; Hugenholtz, P; Blackall, L L; Petray, S; Moss, S; Assis, R A; Fernandez Miyakawa, M; Carloni, G
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.
Hoover, Clare E.; Davenport, Kristen A.; Henderson, Davin M.; Pulscher, Laura A.; Mathiason, Candace K.; Zabel, Mark D.; Hoover, Edward A.
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
Shen, Kaini; Sun, Jian; Cao, Xinxin; Zhou, Daobin; Li, Jian
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.
Barbosa-Solomieu, V; Miossec, L; Vázquez-Juárez, R; Ascencio-Valle, F; Renault, T
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.
Alvarez-Aldana, Adalucy; Martínez, José William; Sepúlveda-Arias, Juan C
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.
Isidro, Raymond A; Isidro, Angel A; Cruz, Myrella L; Hernandez, Siomara; Appleyard, Caroline B
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.
Kajimura, Junko; Ito, Reiko; Manley, Nancy R.; Hale, Laura P.
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
Perizzolo, Marco; Winkfein, Bob; Hui, Susan; Krulicki, Wally; Chan, Jennifer A; Demetrick, Douglas J
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.
AZIMI, ALI; L. KAUFMAN, KIMBERLEY; ALI, MARINA; KOSSARD, STEVEN; FERNANDEZ-PENAS, PABLO
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
Cheng, Jun; He, Jun; Liu, Huaping; Cai, Hao; Hong, Guini; Zhang, Jiahui; Li, Na; Ao, Lu; Guo, Zheng
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
Panigrahi, Manoj Kumar; Suryavanshi, Moushumi; Mehta, Anurag; Saikia, Kandarpa Kumar
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
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
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.
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
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
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
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.
Staff, Synnöve; Kujala, Paula; Karhu, Ritva; Rökman, Annika; Ilvesaro, Joanna; Kares, Saara; Isola, Jorma
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.
Hizem, A; M'rad, S; Oudni-M'rad, M; Mestiri, S; Hammedi, F; Mezhoud, H; Zakhama, A; Mokni, M; Babba, H
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.
Moens, Lotte N J; Falk-Sörqvist, Elin; Ljungström, Viktor; Mattsson, Johanna; Sundström, Magnus; La Fleur, Linnéa; Mathot, Lucy; Micke, Patrick; Nilsson, Mats; Botling, Johan
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.
Gyongyosi, Adrienn; Docs, Otto; Czimmerer, Zsolt; Orosz, Laszlo; Horvath, Attila; Török, Olga; Mehes, Gabor; Nagy, Laszlo; Balint, Balint L
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.
Kajimura, Junko; Ito, Reiko; Manley, Nancy R; Hale, Laura P
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.
Thompson, Seonaid M; Craven, Rachel A; Nirmalan, Niroshini J; Harnden, Patricia; Selby, Peter J; Banks, Rosamonde E
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.
Yakoob, Mohammad Yawar; Hussainy, Akbar Shah
The aim of this study was to observe the histological features of chronic gastritis and associated effects due to Helicobacter pylori infection in 176 randomly selected antral biopsy specimens of chronic gastritis cases. The specimens were reviewed for the presence or absence of H.pylori. The activity (neutrophilic infiltration) of gastritis and the presence or absence of mucosa-associated lymphoid tissue (MALT) were also noted. Chi-square test (Pearson value) was used to analyze categorical variables. H.pylori was detected in 110 (62.5%) cases of chronic gastritis. There was a significant association between H.pylori infection and activity of chronic gastritis (p=0.002). Lymphoid aggregates were significantly more frequently noted in H.pylori-positive patients (68.2%) vs. H.pylori negative group (47%), (p=0.005). It is concluded that H.pylori is significantly associated with active chronic gastritis and with formation of mucosa-associated lymphoid tissue (MALT), which may develop into gastric lymphoma (MALT type).
Ovesen, Sandra; Kirk, Karina Frahm; Nielsen, Hans Linde; Nielsen, Henrik
Campylobacter concisus is an emerging pathogen associated with gastrointestinal disorders such as gastroenteritis and inflammatory bowel diseases (IBD), but the species is also found in healthy subjects. The heterogeneous genome of C. concisus increases the likelihood of varying virulence between strains. Flagella motility is a crucial virulence factor for the well-recognized Campylobacter jejuni; therefore, this study aimed to analyze the motility of C. concisus isolated from saliva, gut biopsies, and feces of patients with IBD, gastroenteritis, and healthy subjects. The motility zones of 63 isolates from 52 patients were measured after microaerobic growth in soft-agar plates for 72 hours. The motility of C. concisus was significantly lower than that of Campylobacter jejuni and Campylobacter fetus subsp. fetus. The motility of C. concisus varied between isolates (4-22 mm), but there was no statistical significant difference between isolates from IBD patients and healthy subjects (p = 0.14). A tendency of a larger motility zones was observed for IBD gut mucosa isolates, although it did not reach statistical significance (p = 0.13), and no difference was found between oral or fecal isolates between groups. In conclusion, the varying motility of C. concisus could not be related to disease outcome or colonization sites.
Yamasaki, Masahiro; Kobayashi, Yusuke; Nakamura, Kensuke; Sasaki, Noboru; Murakami, Masahiro; Rajapakshage, Bandula Kumara Wickramasekara; Ohta, Hiroshi; Yamato, Osamu; Maede, Yoshimitsu; Takiguchi, Mitsuyoshi
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.
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
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
Rothenberger, Meghan K.; Mutuluuza, C. Kityo; Ssali, F.; Jasurda, Jake; Schmidt, Thomas; Schacker, Timothy W.; Beilman, Greg J.
Abstract Background: Lymph nodes and gut-associated lymphatic tissue are important reservoirs of the human immunodeficiency virus (HIV). Little is known about these reservoirs in different geographic populations. We report the surgical outcomes of excisional lymph node and anorectal mucosal biopsies performed internationally and describe the lessons learned. Methods: Patients were recruited through the Joint Clinical Research Center (JCRC) in Kampala, Uganda, where procedures were performed. Studies were approved by the Institutional Review Boards of the JCRC and the University of Minnesota. Instruments and supplies were shipped to Uganda and prepared onsite. Drugs and skin preparations were purchased locally. Lymph nodes were removed through 1–3 cm incisions with ligatures on lymphovascular pedicles. Incisions were closed with subcuticular sutures and epidermal tape. Two to four pieces of anorectal mucosa were obtained through anoscopes using biopsy forceps. Results: One hundred thirty-eight lymph node biopsies and 98 anorectal mucosal biopsies were performed on 71 patients. Forty-one patients were HIV-positive. Many patients had multiple procedures. Two minor complications resulted: One hematoma and one lymphocele. Despite the cost of travel and lodging, cost per biopsy was lower in Uganda compared with the United States. Conclusion: Invasive clinical research can be performed with minimal morbidity in emerging nations with outcomes similar to those found in the United States, but with lower cost. PMID:25650809
Paulsen, I.M.S.; Dimke, H.
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
Pan, Jie; Thoeni, Cornelia; Muise, Aleixo; Yeger, Herman; Cutz, Ernest
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
Hester, Susan D; Bhat, Virunya; Chorley, Brian N; Carswell, Gleta; Jones, Wendell; Wehmas, Leah C; Wood, Charles E
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.
Abdoli, Amir; Dalimi, Abdolhossein; Soltanghoraee, Haleh; Ghaffarifar, Fatemeh
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
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.
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.
Kwon, Mi Jeong; Kim, Ryong Nam; Kim, Yu Jin; Song, Ji-Young; Jung, Kyung Soo; Shin, Young Kee
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
Narayanappa, Rajeswari; Rout, Pritilata; Aithal, Madhuri G S; Chand, Ashis Kumar
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.
Hofbauer, G. F.; Schaefer, C.; Noppen, C.; Böni, R.; Kamarashev, J.; Nestle, F. O.; Spagnoli, G. C.; Dummer, R.
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
Kalmár, Alexandra; Péterfia, Bálint; Hollósi, Péter; Wichmann, Barnabás; Bodor, András; Patai, Árpád V; Schöller, Andrea; Krenács, Tibor; Tulassay, Zsolt; Molnár, Béla
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.
Culpin, Rachel Emily; Sieniawski, Michal; Proctor, Stephen John; Menon, Geetha; Mainou-Fowler, Tryfonia
Tissue biopsy specimens in the form of formalin-fixed paraffin-embedded tissue (FFPET) represent a valuable resource for biomarker identification and validation. However, to date, they remain an underused asset due to uncertainty regarding RNA extraction and the reliability of downstream techniques, including quantitative RT-PCR. Recently, much interest has emerged in the study of microRNAs; small single-stranded RNAs with a role in transcriptional regulation, that are thought to be well preserved in FFPET. In this study, we show that microRNA expression is comparable between FFPET and matched fresh-frozen samples (miR-17-5p: p=0.01, miR-92: p=0.003), and demonstrate that no significant deterioration in expression occurs over prolonged FFPET storage (p=0.06). Furthermore, microRNA expression is equivalent dependant on RNA extraction method (p<0.001) or DNAse treatment of total RNA (p<0.001). Finally, we validate miR-24 as a suitable reference microRNA for diffuse large B-cell lymphoma (DLBCL) FFPET studies.
... Results Mean Cancer Infections, such as tuberculosis Necrotizing granuloma , a type of tumor Nasal polyps Nasal tumors ... Granulomatosis with polyangiitis Juvenile angiofibroma Nasal polyps Necrotizing granuloma Tumor Review Date 4/11/2015 Updated by: ...
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
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
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
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
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
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
Tewari, Deepanker; Kim, Hyun; Feria, Willard; Russo, Brigite; Acland, Helen
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.
Bruinen, Anne L.; van Oevelen, Cateau; Eijkel, Gert B.; Van Heerden, Marjolein; Cuyckens, Filip; Heeren, Ron M. A.
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.
Meksiarun, Phiranuphon; Ishigaki, Mika; Huck-Pezzei, Verena A. C.; Huck, Christian W.; Wongravee, Kanet; Sato, Hidetoshi; Ozaki, Yukihiro
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.
Sidorova, Julia V; Biderman, Bella V; Nikulina, Elena E; Sudarikov, Andrey B
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.
Meksiarun, Phiranuphon; Ishigaki, Mika; Huck-Pezzei, Verena A.C.; Huck, Christian W.; Wongravee, Kanet; Sato, Hidetoshi; Ozaki, Yukihiro
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
Howe, J R; Klimstra, D S; Cordon-Cardo, C
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.
Korabiowska, Monika; Cordon-Cardo, Carlos; Jaenckel, Fredericke; Stachura, Jerzy; Fischer, Gösta; Brinck, Ulrich
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.
Monteith, C E; Chelack, B J; Davis, W C; Haines, D M
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
Bruinen, Anne L; van Oevelen, Cateau; Eijkel, Gert B; Van Heerden, Marjolein; Cuyckens, Filip; Heeren, Ron M A
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.
Rostami, Sima; Torbaghan, Shams Shariat; Dabiri, Shahriar; Babaei, Zahra; Mohammadi, Mohammad Ali; Sharbatkhori, Mitra; Harandi, Majid Fasihi
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
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
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.
Opriessnig, Tanja; Bender, Joseph S; Halbur, Patrick G
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.
Meksiarun, Phiranuphon; Ishigaki, Mika; Huck-Pezzei, Verena A C; Huck, Christian W; Wongravee, Kanet; Sato, Hidetoshi; Ozaki, Yukihiro
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.
Pérez, Cristina; Klaustermeier, Jo Ellen; Alemany, Laia; Tous, Sara; de Sanjosé, Silvia; Velasco, Julio
The great interest in molecular epidemiology of human papilloma virus (HPV) in cervical cancer led us to perform a thorough evaluation of 2 polymerase chain reaction (PCR)-based methods for the detection of HPV in archival formalin-fixed paraffin-embedded (FFPE) samples. Thus, the aim of this study was to compare HPV detection in FFPE samples that have histopathologic diagnosis of invasive cervical cancer using SPF10 broad-spectrum primers PCR followed by DNA enzyme immunoassay and LiPA25 (version 1: Labo Biomedical products, Rijswijk, The Netherlands version 1) and the Papillomavirus Clinical Arrays technique (Genómica, Tres Cantos, Madrid, Spain). In this study, 235 biopsies with histopathologic diagnosis of invasive cervical cancer were analyzed for the detection and genotyping of HPV by LiPA25 SPF10-PCR System (version 1) and Papillomavirus Clinical Arrays technique. The detection of HPV DNA with Genómica technique was 75.1%, and 91.9% with LiPA25 SPF10-PCR. The Genómica technique detected a higher percentage of multiple infections (35%) than LiPA25 (8.9%), with a very low agreement for the detection of multiple infections between them (P>0.05). Our study highlights an important difference between 2 PCR-based methods for detection and genotyping of HPV. LiPA25 SPF10-PCR technology may be more adequate than Genómica for the detection of HPV DNA when using FFPE tissue.
El-Salhy, Magdy; Wendelbo, Ingvild Haukaas; Gundersen, Doris; Hatlebakk, Jan Gunnar; Hausken, Trygve
Animal models are required for research regarding the pathogenesis and efficacy of anti-inflammatory agents in inflammatory bowel disease (IBD). Trinitrobenzene sulfonic acid (TNBS)-induced colitis closely mimics Crohn's disease. The present study was undertaken in order to determine the reliability of following the inflammatory course of TNBS-induced colitis using colonoscopy together with biopsy samples obtained during the examination. In this study we used 20 adult male Wistar rats, with a mean weight of 201.9 g. The rats were divided into two groups, control and TNBS, with ten rats in each group. Following the induction of TNBS colitis, the rats underwent colonoscopy with mucosal biopsies. At the end of the experiment, the rats were sacrificed and whole-wall colonic samples were obtained. The degree of inflammation was assessed endoscopically, macroscopically and microscopically. There was no significant change in the body weight of the control group but significant weight loss was observed in the TNBS group. Examination of the control group did not reveal any inflammation. Severe colitis was observed in the TNBS-induced colitis rats, as assessed endoscopically, macroscopically and microscopically. The endoscopic inflammation score obtained through colonoscopy examinations correlated with that obtained macroscopically, and those obtained microscopically from the whole-wall colon and biopsy samples collected during the colonoscopy. Moreover, the inflammation scores obtained from the whole-wall colon and biopsy samples collected during colonoscopy correlated markedly. In conclusion, colonoscopy is a reliable method for following up the course of inflammation in experimentally induced colitis. Although biopsy samples collected during colonoscopies may be used to assess the degree of inflammation, whole-wall samples are superior in this regard.
Zeng, Yiping; Wang, Fenghua; Williams, Elizabeth D; Chow, Chung Wo
Primary intestinal lymphangiectasia and intestinal lymphatic hypoplasia are 2 causes of protein-losing enteropathy in children and share many common clinical features. For the diagnosis of lymphatic hypoplasia on endoscopic biopsies of the intestine, i.e., based on a negative finding in a small specimen, a very sensitive and specific method for identifying lymphatics is essential. In the present study, lymphatic vessels were labelled using D2-40 immunostaining in mucosal biopsy specimens of the alimentary tract of children in whom no histologic abnormality was noted and of those who had different relatively common pediatric conditions, including inflammatory and neoplastic diseases. Using this method, lymphatic vessels were well visualized even in young infants and not destroyed by diseases. The presence of the muscularis mucosae in specimens was important for adequate assessment. In the duodenum and esophagus, lymphatics were observed in every single section; in the stomach, ileum, and colon, they were less regular and several sections were sometimes required. The extreme sensitivity of this method for demonstrating lymphatic vessels in the duodenum makes it ideal for the histologic diagnosis of intestinal lymphatic hypoplasia. In 4 patients who were considered to have this diagnosis based on clinical features, full-thickness intestinal biopsies and electron microscopy, D2-40 immunostaining confirmed the absence or marked paucity of lymphatics.
Giraudo Conesa, L C; Vannelli, S A; Uzal, F A
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.
Burack, W. Richard; Laughlin, Todd S.; Friedberg, Jonathan; Spence, Janice M.; Rothberg, Paul G.
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
Kondrashova, Olga; Love, Clare J.; Lunke, Sebastian; Hsu, Arthur L.; Waring, Paul M.; Taylor, Graham R.
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
Mondon, J A; Howitt, J; Tosiano, M; Kwok, K W H; Hinton, D E
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.
Korabiowska, Monika; Bauer, Hanne; Quentin, Tomas; Stachura, Jerzy; Cordon-Cardo, Carlos; Brinck, Ulrich
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.
Lo Vasco, Vincenza Rita; Polonia, Patrizia
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.
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
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.
Herraez-Hernandez, Elsa; Preda, Ovidiu; Alonso, Sonia; Pardo, Rosario Serrano; Olmo, Asuncion
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.
Cao, Weisheng; Mays, Jody; Dunn, John; Fulton, Richard; Silva, Robert; Fadly, Aly
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.
Parafioriti, Antonina; Bason, Caterina; Armiraglio, Elisabetta; Calciano, Lucia; Daolio, Primo Andrea; Berardocco, Martina; Di Bernardo, Andrea; Colosimo, Alessia; Luksch, Roberto; Berardi, Anna C.
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
Sánchez, G F D; Banda, R V M; Sahagun, R A; Ledesma, M N; Morales, S E
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.
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
Nuovo, Allison J; Garofalo, Michela; Mikhail, Alexandria; Nicol, Alcina F; Vianna-Andrade, Cecilia; Nuovo, Gerard J
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.
Halldórsdóttir, Anna Margrét; Zehnbauer, Barbara A; Burack, W Richard
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.
Racsa, Lori D; DeLeon-Carnes, Marlene; Hiskey, Matthew; Guarner, Jeannette
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.
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
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.
Nirmalan, Niroshini J; Hughes, Christopher; Peng, Jianhe; McKenna, Therese; Langridge, James; Cairns, David A; Harnden, Patricia; Selby, Peter J; Banks, Rosamonde E
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.
Broeckx, Valérie; Boonen, Kurt; Pringels, Lentel; Sagaert, Xavier; Prenen, Hans; Landuyt, Bart; Schoofs, Liliane; Maes, Evelyne
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.
Li, Jinghuan; Smyth, Paul; Cahill, Susanne; Denning, Karen; Flavin, Richard; Aherne, Sinead; Pirotta, Marco; Guenther, Simone M; O'Leary, John J; Sheils, Orla
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
Dowling, Paul; Moran, Benvon; McAuley, Edel; Meleady, Paula; Henry, Michael; Clynes, Martin; McMenamin, Mairin; Leonard, Niamh; Monks, Mary; Wynne, Bairbre; Ormond, Patrick; Larkin, Annemarie
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
Mubemba, B; Thompson, P N; Odendaal, L; Coetzee, P; Venter, E H
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.
Rossi Spadafora, Marcello Salvatore; Céspedes, Ghislaine; Romero, Sandra; Fuentes, Isabel; Boada-Sucre, Alpidio A.; Cañavate, Carmen; Flores-Chávez, María
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
Uhde, Ann-Kathrin; Lehmbecker, Annika; Baumgärtner, Wolfgang; Spitzbarth, Ingo
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.
Pu, Tianjie; Guo, Peng; Qiu, Yan; Chen, Shinan; Yang, Libo; Sun, Linyong; Ye, Feng; Bu, Hong
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.
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
Lüder Ripoli, Florenza; Mohr, Annika; Conradine Hammer, Susanne; Willenbrock, Saskia; Hewicker-Trautwein, Marion; Hennecke, Silvia; Murua Escobar, Hugo; Nolte, Ingo
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
Ballester, M; Galindo-Cardiel, I; Gallardo, C; Argilaguet, J M; Segalés, J; Rodríguez, J M; Rodríguez, F
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.
Cabaret, Odile; Toussain, Guillaume; Abermil, Nassera; Alsamad, Issam Abd; Botterel, Françoise; Costa, Jean-Marc; Papon, Jean-François; Bretagne, Stéphane
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.
Espinet, Blanca; Bellosillo, Beatriz; Melero, Carme; Vela, Ma Carmen; Pedro, Carmen; Salido, Marta; Pijuan, Lara; Florensa, Lourdes; Besses, Carles; Serrano, Sergi; Solé, Francesc
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.
Taga, Masataka; Eguchi, Hidetaka; Shinohara, Tomoko; Takahashi, Keiko; Ito, Reiko; Yasui, Wataru; Nakachi, Kei; Kusunoki, Yoichiro; Hamatani, Kiyohiro
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.
Yun, Byeong Hwa; Yao, Lihua; Jelaković, Bojan; Nikolić, Jovan; Dickman, Kathleen G; Grollman, Arthur P; Rosenquist, Thomas A; Turesky, Robert J
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.
Van Rossen, Elke; Vander Borght, Sara; van Grunsven, Leo Adrianus; Reynaert, Hendrik; Bruggeman, Veerle; Blomhoff, Rune; Roskams, Tania; Geerts, Albert
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.
Barcelos, Denise; Franco, Marcello F; Leão, Sylvia Cardoso
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.
Luebker, Stephen A; Wojtkiewicz, Melinda; Koepsell, Scott A
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.
Lesnikova, Iana; Lidang, Marianne; Hamilton-Dutoit, Steven; Koch, Jørn
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.
Lass, Ulrike; Hartmann, Christian; Capper, David; Herold-Mende, Christel; von Deimling, Andreas; Meiboom, Maren; Mueller, Wolf
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.
Komminoth, P.; Kunz, E.; Hiort, O.; Schröder, S.; Matias-Guiu, X.; Christiansen, G.; Roth, J.; Heitz, P. U.
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
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
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
Powers, Thomas W; Neely, Benjamin A; Shao, Yuan; Tang, Huiyuan; Troyer, Dean A; Mehta, Anand S; Haab, Brian B; Drake, Richard R
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.
Lai, Xianyin; Schneider, Bryan P
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.
Obersteller, Sonja; Neubauer, Heinrich; Hagen, Ralf Matthias; Frickmann, Hagen
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.
Mansour, Anthony G; Khalil, Pamela Abou; Bejjani, Noha; Chatila, Rajaa; Dagher-Hamalian, Carole; Faour, Wissam H
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.
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.
Wiedorn, Klaus Hermann; Olert, Jürgen; Stacy, Robin A P; Goldmann, Torsten; Kühl, Heike; Matthus, Jutta; Vollmer, Ekkehard; Bosse, Alexander
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.
Ferrari, H F; Luvizotto, M C R; Rahal, P; Cardoso, T C
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.
Sarafanov, A.G.; Todorov, T.I.; Kajdacsy-Balla, A.; Gray, Michael A.; MacIas, V.; Centeno, J.A.
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.
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
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
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
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
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
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.
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
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.
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
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.
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
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.
Lehmann, Annika; Schewe, Christiane; Hennig, Guido; Denkert, Carsten; Weichert, Wilko; Budczies, Jan; Dietel, Manfred
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.
Hosein, Abdel Nasser; Song, Sarah; McCart Reed, Amy E; Jayanthan, Janani; Reid, Lynne E; Kutasovic, Jamie R; Cummings, Margaret C; Waddell, Nic; Lakhani, Sunil R; Chenevix-Trench, Georgia; Simpson, Peter T
Pathology archives contain vast resources of clinical material in the form of formalin-fixed paraffin-embedded (FFPE) tissue samples. Owing to the methods of tissue fixation and storage, the integrity of DNA and RNA available from FFPE tissue is compromized, which means obtaining informative data regarding epigenetic, genomic, and expression alterations can be challenging. Here, we have investigated the utility of repairing damaged DNA derived from FFPE tumors prior to single-nucleotide polymorphism (SNP) arrays for whole-genome DNA copy number analysis. DNA was extracted from FFPE samples spanning five decades, involving tumor material obtained from surgical specimens and postmortems. Various aspects of the protocol were assessed, including the method of DNA extraction, the role of Quality Control quantitative PCR (qPCR) in predicting sample success, and the effect of DNA restoration on assay performance, data quality, and the prediction of copy number aberrations (CNAs). DNA that had undergone the repair process yielded higher SNP call rates, reduced log R ratio variance, and improved calling of CNAs compared with matched FFPE DNA not subjected to repair. Reproducible mapping of genomic break points and detection of focal CNAs representing high-level gains and homozygous deletions (HD) were possible, even on autopsy material obtained in 1974. For example, DNA amplifications at the ERBB2 and EGFR gene loci and a HD mapping to 13q14.2 were validated using immunohistochemistry, in situ hybridization, and qPCR. The power of SNP arrays lies in the detection of allele-specific aberrations; however, this aspect of the analysis remains challenging, particularly in the distinction between loss of heterozygosity (LOH) and copy neutral LOH. In summary, attempting to repair DNA that is damaged during fixation and storage may be a useful pretreatment step for genomic studies of large archival FFPE cohorts with long-term follow-up or for understanding rare cancer types, where
Salawu, Abdulazeez; Ul-Hassan, Aliya; Hammond, David; Fernando, Malee; Reed, Malcolm; Sisley, Karen
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.
Tanic, Miljana; Yanowski, Kira; Gómez-López, Gonzalo; Rodriguez-Pinilla, María Socorro; Marquez-Rodas, Iván; Osorio, Ana; Pisano, David G; Martinez-Delgado, Beatriz; Benítez, Javier
Screening for germline mutations in breast cancer-associated genes BRCA1 and BRCA2 is indicated for patients with breast cancer from high-risk breast cancer families and influences both treatment options and clinical management. However, only 25% of selected patients test positive for BRCA1/2 mutation, indicating that additional diagnostic biomarkers are necessary. We analyzed 124 formalin-fixed paraffin-embedded (FFPE) tumor samples from patients with hereditary (104) and sporadic (20) invasive breast cancer, divided into two series (A and B). Microarray expression profiling of 829 human miRNAs was performed on 76 samples (Series A), and bioinformatics tool Prophet was used to develop and test a microarray classifier. Samples were stratified into a training set (n = 38) for microarray classifier generation and a test set (n = 38) for signature validation. A 35-miRNA microarray classifier was generated for the prediction of BRCA1/2 mutation status with a reported 95% (95% CI = 0.88-1.0) and 92% (95% CI: 0.84-1.0) accuracy in the training and the test set, respectively. Differential expression of 12 miRNAs between BRCA1/2 mutation carriers versus noncarriers was validated by qPCR in an independent tumor series B (n = 48). Logistic regression model based on the expression of six miRNAs (miR-142-3p, miR-505*, miR-1248, miR-181a-2*, miR-25* and miR-340*) discriminated between tumors from BRCA1/2 mutation carriers and noncarriers with 92% (95% CI: 0.84-0.99) accuracy. In conclusion, we identified miRNA expression signatures predictive of BRCA1/2 mutation status in routinely available FFPE breast tumor samples, which may be useful to complement current patient selection criteria for gene testing by identifying individuals with high likelihood of being BRCA1/2 mutation carriers.
Everest-Dass, Arun V; Briggs, Matthew T; Kaur, Gurjeet; Oehler, Martin K; Hoffmann, Peter; Packer, Nicolle H
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
Kadota, Kyuichi; Kannisto, Eric; Jones, David R.; Adusumilli, Prasad S.
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
Background Epidemiological evidence links Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) to B-cell non-Hodgkin lymphoma (B-NHL). These B-NHLs, particularly those associated with HCV, may represent a distinct sub-group with peculiar molecular features, including peculiar expression of microRNAs (miRs). The aim of the present study was to search for miRs whose level in indolent B-NHL tissues could be associated with HBV or HCV infection. Methods Fourteen formalin fixed paraffin embedded (FFPE) tissues from HBV+, HCV+ and HBV-/HCV- indolent B-NHL patients were analyzed for levels of 34 selected miRs by quantitative Real-Time PCR. Reactive lymph nodes (RLNs) from HBV-/HCV- patients were included as non-tumor control. Statistical analysis of output data included Pearson and Spearman correlation and Mann-Whitney test and were carried out by the STATA software. Results MiR-92a was decreased exclusively in HBV-/HCV- B-NHLs, while miR-30b was increased in HBV+ and HCV+ samples, though only the HCV+ achieved full statistical significance. Analysis of a small subset of B-NHLs belonging to the same histological subtype (Nodal Marginal Zone Lymphoma) highlighted three miRs associated with HCV infection (miR-223, miR-29a and miR-29b) and confirmed decreased level of miR-92a in HBV-/HCV- samples also when considering this restricted B-NHL group. Conclusions Although caution is needed due to the limited number of analyzed samples, overall the results suggest that differences at the miR expression level exist between indolent B-NHLs developed in patients with or without HBV or HCV infection. The identification of three further miRs associated with HCV by analyzing histologically homogeneous samples suggests that variations of miR levels possibly associated with HBV or HCV may be obscured by the tissue-specific variability of miR level associated with the different histological subtypes of B-NHL. Thus, the identification of further miRs will require, in addition to an increased
Coamplification at lower denaturation temperature polymerase chain reaction enables selective identification of K-Ras mutations in formalin-fixed, paraffin-embedded tumor tissues without tumor-cell enrichment.
Yu, Shaorong; Xie, Li; Hou, Zhibo; Qian, Xiaoping; Yu, Lixia; Wei, Jia; Ding, Yitao; Liu, Baorui
Conventional polymerase chain reaction-based Sanger sequencing is the standard assay for the detection of K-Ras mutations. However, this method is deficient in identifying small numbers of mutation-bearing cells, and tumor-cell enrichment methods such as microdissection or macrodissection are labor intensive and not always achievable. We applied the recently described coamplification at lower denaturation temperature polymerase chain reaction, which amplifies minority alleles selectively, to detect K-Ras mutations directly in 29 formalin-fixed, paraffin-embedded pancreatic specimens and compared the results with those of conventional polymerase chain reaction. To avoid a false-negative result from the coamplification at lower denaturation temperature polymerase chain reaction assay, we applied a more sensitive peptide nucleic acid polymerase chain reaction method as the gold standard. Dilution experiments indicated an approximately 5-fold improvement in sensitivity with coamplification at lower denaturation temperature polymerase chain reaction-based Sanger sequencing. Conventional polymerase chain reaction detected K-Ras mutations in 11 formalin-fixed, paraffin-embedded pancreatic specimens (37.9%), whereas coamplification at lower denaturation temperature polymerase chain reaction could identify all of those mutations as well as mutations in 10 additional samples, for a total of 21 (72.4%, P = .002) of 29. Unlike peptide nucleic acid polymerase chain reaction, coamplification at lower denaturation temperature polymerase chain reaction identified all K-Ras mutations in specimens in which tumor cells accounted for at least 20% of the total. Adoption of coamplification at lower denaturation temperature polymerase chain reaction is straightforward and requires no additional reagents or instruments. The technique is a good strategy to detect K-Ras mutations selectively in formalin-fixed, paraffin-embedded tissues without tumor-cell enrichment.
Miyahara, Glauco Issamu; Simonato, Luciana Estevam; Mattar, Neivio José; Camilo Jr, Deolino João; Biasoli, Eder Ricardo
The purpose of this study was to compare the histopathological analysis with polymerase chain reaction (PCR) methods to predict the presence of human papillomavirus (HPV) infection in oral squamous cell carcinoma biopsies. Eighty-three paraffin-embedded tissue specimens from patients with oropharynx and mouth floor squamous cell carcinoma were submitted to histopathological analysis under light microscopy, specifically for the determination of the presence of koilocytes. Subsequently, DNA was purified from the same paraffin-embedded specimens and submitted to PCR. Fisher's exact test showed no statistically significant correlation between the two methods. The results suggest that the presence of koilocytes is unreliable for the detection of HPV presence in oral and oropharynx squamous cell carcinoma.
Lin, Chun-Ming; Jeng, Chian-Ren; Hsiao, Shih-Hsuan; Chang, Chih-Cheng; Liu, Chen-Hsuan; Tsai, Yi-Chieh; Chia, Mi-Yuan; Pang, Victor Fei
Taking advantage of the high sensitivity of polymerase chain reaction (PCR) and the cell-localizing ability of in situ hybridization (ISH), an indirect in situ PCR (ISPCR) method was developed for detecting the distribution of porcine circovirus type 2 (PCV2) in formalin-fixed and paraffin-embedded inguinal lymph nodes obtained from clinically healthy PCV2-carrier pigs and postweaning multisystemic wasting syndrome (PMWS)-affected pigs. Comparisons of the relative sensitivity of indirect ISPCR with other routinely used diagnostic methods for PCV2 indicated that nested PCR was the most sensitive method followed by indirect ISPCR, conventional PCR, ISH, and immunohistochemical (IHC) staining. Although indirect ISPCR, ISH, and IHC staining all revealed a similar signal distribution pattern of PCV2, using indirect ISPCR allowed specific amplification and detection of previously uneasily detected PCV2 signal than by routine ISH or IHC staining, particularly in those cells within the germinal center in clinically healthy PCV2-carrier pigs. Furthermore, six different PCV2 signal expression patterns in conjunction with the correlated lymphoid lesion stages were classified to describe the tissue morphological changes and viral infection. The result indicates that indirect ISPCR is a more effective, cell-based diagnostic tool with good specificity to detect limited PCV2 infection in formalin-fixed and paraffin-embedded tissue specimens and it would be a useful tool for further exploring the pathogenesis of PCV2 infection.
Yantiss, Rhonda K; Das, Kiron M; Farraye, Francis A; Odze, Robert D
Distinction between ulcerative colitis (UC) and Crohn disease (CD) in mucosal biopsies is often difficult. Das-1 and CG-3 are monoclonal antibodies directed against an unknown colonic epithelial protein and human tropomyosin isoform-5, respectively, both show altered expression in patients with UC. In this study, we evaluated the utility of Das-1 and CG-3 in distinguishing UC from CD and from other types of colitis. One colonic biopsy specimen from each of 85 patients with confirmed UC (n=25), CD (n=15), lymphocytic (n=15), collagenous (n=15), and ischemic (n=15) colitis, and also 10 samples from normal controls, were stained for Das-1 and CG-3 using standard techniques. Reactivity for Das-1 and CG-3 was noted to be absent or present, and the location (ie, surface+/-crypt epithelium) and degree (weak or strong) of CG-3 staining was recorded. Loss of Das-1 staining occurred more frequently in UC (96%) compared with CD (20%), lymphocytic (20%), collagenous (13%), and ischemic colitis (0%) cases, as well as controls (10%, P<0.001 for all comparisons). CG-3 positivity in crypt epithelium was significantly more common in UC (52%) compared with the other groups (P< or =0.02 for all comparisons). The combination of strong crypt CG-3 staining and loss of Das-1 staining was noted in 44% of UC cases, but not in any other type of colitis (P=0.003 for all comparisons). We conclude that the patterns of Das-1 and CG-3 staining in colonic mucosal biopsies may be clinically useful in distinguishing UC from CD and from other colitidies.
Assessment of mutations of Ha- and Ki-ras oncogenes and the p53 suppressor gene in seven malignant mesothelioma patients exposed to asbestos--PCR-SSCP and sequencing analyses of paraffin-embedded primary tumors.
Kitamura, F; Araki, S; Tanigawa, T; Miura, H; Akabane, H; Iwasaki, R
To examine whether malignant mesothelioma due to asbestos has genetic alterations in the Ha- and Ki-ras oncogenes or in the p53 suppressor gene, we analyzed the point mutations of these genes in paraffin-embedded autopsy samples of the primary tumors of malignant mesothelioma in seven asbestos patients who died from malignant mesothelioma. The genetic analysis was conducted by the polymerase chain reaction-single strand comformation polymorphysms (PCR-SSCP) method in all patients, and through the sequencing of deoxyribonucleic acid (DNA) bases in one patient. No genetic alterations were found in exons 1 or 2 of Ha- and Ki-ras oncogenes, or in exons 5 to 9 of the p53 gene, in any of the patients. Further studies on a larger number of patients are required to reach a definite conclusion concerning the genetic effects of asbestos on malignant mesothelioma.
Carbohydrate digesting enzyme activities are measured in duodenal biopsies to detect deficiencies of lactase and sucrase activities, however glucoamylase (GA) assays for starch digestion are not included. Because food starch represents half of energy intake in the human diet, assays for starch diges...
Adam, Benjamin; Mengel, Michael
Despite its long-standing status as the diagnostic "gold standard", the renal transplant biopsy is limited by a fundamental dependence on descriptive, empirically-derived consensus classification. The recent shift towards personalized medicine has resulted in an increased demand for precise, mechanism-based diagnoses, which is not fully met by the contemporary transplantation pathology standard of care. The expectation is that molecular techniques will provide novel pathogenetic insights that will allow for the identification of more accurate diagnostic, prognostic, and therapeutic targets. Here we review the current state of molecular renal transplantation pathology. Despite significant research activity and progress within the field, routine adoption of clinical molecular testing has not yet been achieved. The recent development of novel molecular platforms suitable for use with formalin-fixed paraffin-embedded tissue will offer potential solution for the major barriers to implementation. The recent incorporation of molecular diagnostic criteria into the 2013 Banff classification is a reflection of progress made and future directions in the area of molecular transplantation pathology. Transcripts related to endothelial injury and NK cell activation have consistently been shown to be associated with antibody-mediated rejection. Prospective multicenter validation and implementation of molecular diagnostics for major entities remains an unmet clinical need in transplantation. It is expected that an integrated system of transplantation pathology diagnosis comprising molecular, morphological, serological, and clinical variables will ultimately provide the greatest diagnostic precision.
Spraker, Terry R; VerCauteren, Kurt C; Gidlewski, Tom L; Munger, Randy D; Walter, W David; Balachandran, Aru
To determine if the number of rectal lymphoid follicles decreases with respect to age and sex relative to diagnosis of chronic wasting disease (CWD), rectal biopsies (n = 1,361) were taken from captive Rocky Mountain elk (Cervus elaphus nelsoni) at 4 ranches in the western United States between 2005 and 2008. Rectal tissues were stained with a monoclonal antibody (F99/97.6.1), which selectively stains the abnormal isoform of the prion protein associated with CWD of elk. The number of lymphoid follicles obtained from typical biopsy tissues decreased with the age of the animal. The acceptable number of lymphoid follicles for detection of CWD was not considered to be a problem in elk up to 8.5 years of age, but in elk over 8.5 years of age, the follicle count was considered to be low. Sex of the animal had no effect on the number of lymphoid follicles observed in each age group. Rectal biopsies were an accurate test to diagnose preclinical stages of CWD in elk but may be best suited to elk that are less then 8.5 years of age.
... Physician Resources Professions Site Index A-Z Bone Biopsy Bone biopsy uses a needle and imaging guidance ... limitations of Bone Biopsy? What is a Bone Biopsy? A bone biopsy is an image-guided procedure ...
Renal biopsy; Biopsy - kidney ... Barisoni L, Arend LJ, Thomas DB. Introduction to renal biopsy. In: Zhou M, Mari-Galluzzi C, eds. ... Saunders; 2015:chap 7. Topham PS, Chen Y. Renal biopsy. In: Johnson RJ, Feehally J, Floege J, ...
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
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
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.
Kan, Robert K; Pleva, Christina M; Hamilton, Tracey A; Petrali, John P
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.
The Relationship between the Presence of Chromosomal Instability and Prognosis of Squamous Cell Carcinoma of the Lung: Fluorescence in situ Hybridization Analysis of Paraffin-embedded Tissue from 47 Korean Patients
Yoo, Jung-Wan; Seo, Kwang Won; Jang, Se Jin; Oh, Yeon-Mock; Shim, Tae Sun; Kim, Woo Sung; Lee, Dong-Soon; Lee, Sang-Do
To evaluate the prognostic importance of chromosomal instability (CIN) in squamous cell carcinoma (SCC) of the lung, the relationship between CIN detected by fluorescence in situ hybridization (FISH) and survival in SCC patients was examined. Forty-seven surgical specimens of lung SCC 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 age, T factor, N factor, CIN, and smoking status. A sample was defined as CIN-positive if at least four of the five chromosomes were positive. Among the 47 specimens, 9 (19%) were CIN-positive. The overall survival rate was 66%. Overall survival rates were estimated as 33.3% for CIN-positive patients and 76.7% for CIN-negative patients (Hazard ratio 3.47; 95% Confidence interval, 1.25-9.67; P=0.017). In multivariate analysis, the presence of CIN was a predictive factor for survival. CIN-positive based on FISH can be prognostic factor of lung SCC. PMID:20514306
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
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.
Diagnostic sensitivity and specificity of in situ hybridization and immunohistochemistry for Eastern equine encephalitis virus and West Nile virus in formalin-fixed, paraffin-embedded brain tissue of horses.
Pennick, Kate E; McKnight, Christy A; Patterson, Jon S; Latimer, Kenneth S; Maes, Roger K; Wise, Annabel G; Kiupel, Matti
Immunohistochemistry (IHC) and in situ hybridization (ISH) can be used either to detect or to differentiate between Eastern equine encephalitis virus (EEEV) and West Nile virus (WNV) within formalin-fixed, paraffin-embedded (FFPE) brain tissue of horses. To compare the diagnostic sensitivity and specificity of ISH and IHC, FFPE brain tissue from 20 EEEV-positive horses and 16 WNV-positive horses were tested with both EEEV and WNV oligoprobes and EEEV- and WNV-specific antibodies. Reverse transcription polymerase chain reaction (RT-PCR) for detection of EEEV and WNV was used as the gold standard to confirm infection. All horses that tested positive for EEEV by RT-PCR also tested positive by IHC and ISH, except for 1 case that was false-negative by ISH. In contrast, all horses that tested positive for WNV by RT-PCR tested negative by IHC and only 2 horses tested positive by ISH. No false-positives were detected with either method for both viruses. Both IHC and ISH are highly specific and sensitive diagnostic methods to detect EEEV in equine FFPE brain tissues, although neither appear effective for the diagnosis of WNV in equine neurologic cases.
Nizard, Mevyn; Diniz, Mariana O; Roussel, Helene; Tran, Thi; Ferreira, Luis CS; Badoual, Cecile; Tartour, Eric
The mucosal immune system displays several adaptations reflecting the exposure to the external environment. The efficient induction of mucosal immune responses also requires specific approaches, such as the use of appropriate administration routes and specific adjuvants and/or delivery systems. In contrast to vaccines delivered via parenteral routes, experimental, and clinical evidences demonstrated that mucosal vaccines can efficiently induce local immune responses to pathogens or tumors located at mucosal sites as well as systemic response. At least in part, such features can be explained by the compartmentalization of mucosal B and T cell populations that play important roles in the modulation of local immune responses. In the present review, we discuss molecular and cellular features of the mucosal immune system as well as novel immunization approaches that may lead to the development of innovative and efficient vaccines targeting pathogens and tumors at different mucosal sites. PMID:25424921
Argani, Pedram; Yonescu, Raluca; Morsberger, Laura; Morris, Kerry; Netto, George J; Smith, Nathan; Gonzalez, Nilda; Illei, Peter B; Ladanyi, Marc; Griffin, Constance A
A subset of renal cell carcinomas (RCCs) is characterized by t(6;11)(p21;q12), which results in fusion of the untranslated Alpha (MALAT1) gene to the TFEB gene. Only 21 genetically confirmed cases of t(6;11) RCCs have been reported. This neoplasm typically demonstrates a distinctive biphasic morphology, comprising larger epithelioid cells and smaller cells clustered around basement membrane material; however, the full spectrum of its morphologic appearances is not known. The t(6;11) RCCs differ from most conventional RCCs in that they consistently express melanocytic immunohistochemical (IHC) markers such as HMB45 and Melan A and the cysteine protease cathepsin K but are often negative for epithelial markers such as cytokeratins. TFEB IHC has been proven to be useful to confirm the diagnosis of t(6;11) RCCs in archival material, because native TFEB is upregulated through promoter substitution by the gene fusion. However, IHC is highly fixation dependent and has been proven to be particularly difficult for TFEB. A validated fluorescence in situ hybridization (FISH) assay for molecular confirmation of the t(6;11) RCC in archival formalin-fixed, paraffin-embedded material has not been previously reported. We report herein the development of a break-apart TFEB FISH assay for the diagnosis of t(6;11)(p21;q12) RCCs. We validated the assay on 4 genetically confirmed cases and 76 relevant expected negative control cases and used the assay to report 8 new cases that expand the clinicopathologic spectrum of t(6;11) RCCs. An additional previously reported TFEB IHC-positive case was confirmed by TFEB FISH in 46-year-old archival material. In conclusion, TFEB FISH is a robust, clinically validated assay that can confirm the diagnosis of t(6;11) RCC in archival material and should allow a more comprehensive clinicopathologic delineation of this recently recognized neoplastic entity.
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.
Chaux, Alcides; Cohen, Julie S; Schultz, Luciana; Albadine, Roula; Jadallah, Sana; Murphy, Kathleen M; Sharma, Rajni; Schoenberg, Mark P; Netto, George J
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.
Hormone Receptor Expression Analyses in Neoplastic and Non-Neoplastic Canine Mammary Tissue by a Bead Based Multiplex Branched DNA Assay: A Gene Expression Study in Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Samples
Mohr, Annika; Lüder Ripoli, Florenza; Hammer, Susanne Conradine; Willenbrock, Saskia; Hewicker-Trautwein, Marion; Kiełbowicz, Zdzisław; Murua Escobar, Hugo; Nolte, Ingo
Immunohistochemistry (IHC) is currently considered the method of choice for steroid hormone receptor status evaluation in human breast cancer and, therefore, it is commonly utilized for assessing canine mammary tumors. In case of low hormone receptor expression, IHC is limited and thus is complemented by molecular analyses. In the present study, a multiplex bDNA assay was evaluated as a method for hormone receptor gene expression detection in canine mammary tissues. Estrogen receptor (ESR1), progesterone receptor (PGR), prolactin receptor (PRLR) and growth hormone receptor (GHR) gene expressions were evaluated in neoplastic and non-neoplastic canine mammary tissues. A set of 119 fresh frozen and 180 formalin-fixed, paraffin-embedded (FFPE) was comparatively analyzed and used for assay evaluation. Furthermore, a possible association between the hormone receptor expression in different histological subtypes of canine malignant mammary tumors and the castration status, breed and invasive growth of the tumor were analyzed. The multiplex bDNA assay proved to be more sensitive for fresh frozen specimens. Hormone receptor expression found was significantly decreased in malignant mammary tumors in comparison to non-neoplastic tissue and benign mammary tumors. Among the histological subtypes the lowest gene expression levels of ESR1, PGR and PRLR were found in solid, anaplastic and ductal carcinomas. In summary, the evaluation showed that the measurement of hormone receptors with the multiplex bDNA assay represents a practicable method for obtaining detailed quantitative information about gene expression in canine mammary tissue for future studies. Still, comparison with IHC or quantitative real-time PCR is needed for further validation of the present method. PMID:27649560
Hillig, Thore; Thode, Jørgen; Breinholt, Marie F; Franzmann, Maria-Benedicte; Pedersen, Carsten; Lund, Flemming; Mygind, Henrik; Sölétormos, György; Rudnicki, Martin
We compare HER2 receptor amplification analysis by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and real-time polymerase chain reaction (real-time PCR) DNA copy-number assay following laser capture microdissection (LCM) in formalin-fixed paraffin embedded tissue from 40 women with verified ovarian cancer. We speculate that LCM should result in a more accurate assessment of HER2 amplification in our real-time PCR assay compared with IHC and FISH. HER2 overexpression measured by IHC, FISH, or real-time PCR was found in 5.0%, 5.0%, and 22.5%, respectively. HER2 negative results measured by IHC, FISH, or real-time PCR were found in 95%, 92.5%, and 60.0%, respectively. Analysis failed for IHC, FISH, or real-time PCR in 0%, 2.5%, or 17.5% of cases. Concordance between IHC and FISH, IHC and real-time PCR, or FISH and real-time PCR were 89.7%, 72.7%, or 78.1%, respectively. Only few ovarian cancer patients were HER2 overexpressed measured by IHC or FISH and thus could be eligible for antibody-based therapy with trastuzumab (Herceptin). Interestingly, we find an increased number of HER2 positive patients by real-time PCR analysis on microdissected cancer cells, suggesting a number of HER2 positive patients not detected by current methods. Thus, the concept of quantitative measurement of HER2 on microdissected cancer cells should be explored further.
Solassol, Jérôme; Vendrell, Julie; Märkl, Bruno; Haas, Christian; Bellosillo, Beatriz; Montagut, Clara; Smith, Matthew; O’Sullivan, Brendan; D’Haene, Nicky; Le Mercier, Marie; Grauslund, Morten; Melchior, Linea Cecilie; Burt, Emma; Cotter, Finbarr; Stieber, Daniel; Schmitt, Fernando de Lander; Motta, Valentina; Lauricella, Calogero; Colling, Richard; Soilleux, Elizabeth; Fassan, Matteo; Mescoli, Claudia; Collin, Christine; Pagès, Jean-Christophe; Sillekens, Peter
Since the advent of monoclonal antibodies against epidermal growth factor receptor (EGFR) in colorectal cancer therapy, the determination of RAS mutational status is needed for therapeutic decision-making. Most prevalent in colorectal cancer are KRAS exon 2 mutations (40% prevalence); lower prevalence is observed for KRAS exon 3 and 4 mutations (6%) and NRAS exon 2, 3, and 4 mutations (5%). The Idylla™ KRAS Mutation Test on the molecular diagnostics Idylla™ platform is a simple (<2 minutes hands-on time), highly reliable, and rapid (approximately 2 hours turnaround time) in vitro diagnostic sample-to-result solution. This test enables qualitative detection of 21 mutations in codons 12, 13, 59, 61, 117, and 146 of the KRAS oncogene being clinically relevant according to the latest clinical guidelines. Here, the performance of the Idylla™ KRAS Mutation Assay, for Research Use Only, was assessed on archived formalin-fixed paraffin-embedded (FFPE) tissue sections by comparing its results with the results previously obtained by routine reference approaches for KRAS genotyping. In case of discordance, samples were assessed further by additional methods. Among the 374 colorectal cancer FFPE samples tested, the overall concordance between the Idylla™ KRAS Mutation Assay and the confirmed reference routine test results was found to be 98.9%. The Idylla™ KRAS Mutation Assay enabled detection of 5 additional KRAS-mutated samples not detected previously with reference methods. As conclusion the Idylla™ KRAS Mutation Test can be applied as routine tool in any clinical setting, without needing molecular infrastructure or expertise, to guide the personalized treatment of colorectal cancer patients. PMID:27685259
In situ hybridization for Coccidioides immitis 5.8S ribosomal RNA sequences in formalin-fixed, paraffin-embedded pulmonary specimens using a locked nucleic acid probe: a rapid means for identification in tissue sections.
Montone, Kathleen T; Litzky, Leslie A; Feldman, Michael D; Peterman, Heather; Mathis, Benjamin; Baliff, Jeffrey; Kaiser, Larry R; Kucharczuk, John; Nachamkin, Irving
Coccidioides immitis/Coccidioides posadasii are common causes of pulmonary infection in certain geographic areas, and are highly infectious when working with culture isolates in the laboratory. Rapid techniques to accurately identify this pathogen in tissues may be of benefit for diagnosis and in limiting the exposure of laboratory personnel to this agent. Locked nucleic acids (LNA) are modified nucleotides in which a ribonucleoside is linked between the 2'-oxygen and the 4'-carbon atoms with a methylene unit. LNA oligonucleotides exhibit increased thermal stability and make excellent probes for in situ hybridization (ISH). In this study, ISH utilizing a biotin-labeled LNA probe targeting Coccidioides sp. ribosomal RNA sequences in 6 formalin-fixed, paraffin-embedded pulmonary tissue specimens from 6 patients with culture positive or histologic findings suggestive of Coccidioides sp. infection is described. The cultures of the pulmonary specimens confirmed C. immitis in 3 of 6 patients. The ISH procedure with the LNA probe was positive in all 6 cases, although the number of organisms that were highlighted varied from rare to numerous. ISH with a biotin-labeled DNA probe of the same sequence was positive in 4 of the 6 cases and the signal intensity and number of organisms was much less than that observed with the LNA probe. Negative control tissues containing a variety of different fungal pathogens including Aspergillus sp., Fusarium sp., Blastomyces dermatitidis, Candida sp, Histoplasma capsulatum, and Zygomyces did not hybridize with the LNA and DNA probes. ISH with an LNA oligonucleotide probe targeting Coccidioides sp. ribosomal RNA is useful for rapid ISH. ISH could be rapidly performed when fungal pathogens are observed in tissue but cultures are negative or have not been performed.
Harlé, Alexandre; Busser, Benoit; Rouyer, Marie; Harter, Valentin; Genin, Pascal; Leroux, Agnès; Merlin, Jean-Louis
Many studies documented the influence of KRAS mutation status on the response of patients with metastatic colorectal cancer (mCRC) to anti-EGFR monoclonal antibodies. The COBAS 4800 KRAS is an assay using real time PCR and TaqMelt technology, CE-IVD validated, for the detection of 19 KRAS somatic mutations in exons 2 and 3. We compared COBAS with previously validated PCR TaqMan and High Resolution Melting (HRM) assays on 156 formalin-fixed paraffin embedded (FFPE) specimens of colorectal carcinoma. DNA extraction procedures, using the Qiagen QiAMP kit and the Roche COBAS DNA kit, were also compared. Of the 156 samples, 132 were interpretable using COBAS and TaqMan and 92 using COBAS and HRM. No statistically significant difference was found between COBAS/TaqMan and COBAS/HRM (k = 0.937; p < 0.001 - four discordant cases were found, mostly concerning codon 61 mutations and k = 0.891; p < 0.001 - five discordant cases were found, three regarding codon 61 and two on codon 12/13, respectively). No difference was found between the two DNA extraction methods (t = 1.7185; dol = 39; α = 5 %). The three assays were found suitable to detect accurately KRAS mutations in colon FFPE specimens. COBAS and TaqMan were found to be more robust than HRM, as they yielded fewer non-interpretable results. DNA extraction kits were found to provide equivalent results. The present study shows that pre-screening using COBAS with further TaqMan mutation characterization constitutes an easy and reliable approach for routine diagnostic purposes.
Nechifor-Boilă, Adela Corina; Loghin, Andrada; Vacariu, Victor; Halaţiu, Vasile Bogdan; Borda, Angela
Optimal recovery of nucleic acids from formalin-fixed paraffin-embedded (FFPE) tissues is highly dependent on a series of pre-extraction steps, mainly related (but not limited) to fixation. The aim of our study was to investigate if the storage period of the FFPE blocks had a significant effect on the isolated DNA. We examined the quantity and purity of the isolated DNA from 83 FFPE blocks, corresponding to malignant thyroid (n=28) and renal (n=55) carcinomas that had been stored in our department for up to eight years. The DNA extraction protocol was based on a precipitation method (MasterPure™ DNA Purification Kit, Epicentre), in accordance to the manufacturer instructions, optimized in our laboratory. A spectrophotometer was used to determine the yield (A260) and purity (A260/A280 ratio) of the isolated DNA. We successfully isolated good DNA quantity and purity from all our study cases (mean concentration: 223.4 ± 104.16 ng/μL; mean A260/A280 ratio: 1.68 ± 0.09). Moreover, no statistically significant differences were observed between tumor blocks stored for 2-3 years and 7-8 years, respectively, both in terms of DNA quantity (p=0.196) and purity (p=0.663). In conclusion, we successfully validated an efficient, reproducible DNA extraction technique that provided a good range of DNA concentrations and purity, regardless the type of tissue (thyroid or kidney). Moreover, we demonstrated that the storage period of the FFPE blocks does not have a significant influence on the DNA quantity and purity.
Polyp biopsy ... are treated is the colon. How a polyp biopsy is done depends on the location: Colonoscopy or flexible sigmoidoscopy explores the large bowel Colposcopy-directed biopsy examines the vagina and cervix Esophagogastroduodenoscopy (EGD) or ...
Biopsy - liver; Percutaneous biopsy ... the biopsy needle to be inserted into the liver. This is often done by using ultrasound. The ... the chance of damage to the lung or liver. The needle is removed quickly. Pressure will be ...
... Series Urinary Tract Imaging Urodynamic Testing Virtual Colonoscopy Liver Biopsy What is a liver biopsy? A liver biopsy is a procedure that ... remove the liver tissue sample. What is the liver and what does it do? The liver is ...
Ballester Sánchez, R; de Unamuno Bustos, B; Navarro Mira, M; Botella Estrada, R
Mucosal melanoma is a rare melanoma subtype that differs from the cutaneous form of the tumor in its biology, clinical manifestations, and management. Diagnosis is usually late due to a lack of early or specific signs and the location of lesions in areas that are difficult to access on physical examination. Surgical excision is the treatment of choice for localized disease. The value of sentinel lymph node biopsy and lymphadenectomy is still unclear. Radiotherapy can be used as adjuvant therapy for the control of local disease. c-KIT mutations are more common than in other types of melanoma and this has led to significant advances in the use of imatinib for the treatment of metastatic mucosal melanoma.
Hieu, Nguyen Minh; Nam, Nguyen Hoang; Huyen, Nguyen Thi; Van Anh, Nguyen Thi; Nghia, Phan Tuan; Khoa, Nguyen Ba; Toan, Nguyen Linh; Luong, Nguyen Hoang
SiO2-coated Fe3O4 nanoparticles (Fe3O4@SiO2 NPs) were successfully synthesized using ultrasound in order to extract DNA from cancer tissues for application in diagnostics. The core 10.7-nm-diameter Fe3O4 nanoparticles were synthesized by co-precipitation of Fe3+ and Fe2+ as reaction substrates and NH4OH as precipitant, then coated with a thin layer of amorphous silica by a modified Stober method. Further SiO2 coating using alkaline hydrolysis of tetraethyl orthosilicate in ethanol and water mixture was accelerated in the presence of a 37-kHz ultrasound, resulting in the NPs having different sizes of 14.5 nm (version M1), 24.4 nm (version M2), and 34.9 nm (version M3) with saturation magnetization values of 50.2 emu/g, 18.6 emu/g, 10.3 emu/g, respectively. Among the three Fe3O4@SiO2 NPs versions, the M1 NPs allowed extraction of DNAs from 10 mg formalin-fixed and paraffin-embedded (FFPE) tissues of nasopharyngeal carcinoma patients with the highest recovery of about 100-500 ng/μl and good purity (A260/A280: 1.8-1.9). The extracted DNAs could be used as templates for downstream amplification of 252-bp sequencing specifically for the Braf cancer biomarker gene using polymerase chain reaction (PCR), as well as detection of the pathogenic Epstein-Barr virus (EBV) and the human papilloma-virus (HPV) using real-time PCR. DNA extraction recoveries of both EBV and HPV using Fe3O4@SiO2 NPs M1 were significantly better that those using commercialized Fe3O4@SiO2 microbeads, as indicated by lower threshold cycles of all fluorescent signals including fluorescein amidite (FAM) dye representative for EBV infection, hexachlorofluorescein (HEX) dye representative for β-globin (internal control), and SYBR Green dye representative for HPV infection in tested clinical samples from patients with nasopharyngeal carcinoma (NPC).
Wang, Hye-Young; Kim, Geehyuk; Cho, Hyemi; Kim, Sunghyun; Lee, Dongsup; Park, Sunyoung; Park, Kwang Hwa; Lee, Hyeyoung
Human papillomavirus (HPV) is a major cause of cervical cancer, which is the third most common cancer in women. Human telomerase reverse transcriptase (hTERT) and Ki67 are tumor cell markers indicating cancer cell proliferation in cancer patients, and activation of hTERT and Ki67 leads to progressive cervical carcinogenesis. In the present study, we evaluated the CervicGen HPVE6/E7 mRNA RT-qDx assay, which detects 16 HPV high-risk (HR) genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 and 69), and the CervicGen hTERT and Ki67 mRNA RT-qDx assay using 117 formalin-fixed paraffin-embedded (FFPE) cervical cancer tissue samples. The diagnostic validity of the CervicGen HPV RT-qDx assay for detecting histologically proven prevalent squamous cell carcinoma (SCC) was 94% sensitivity, 100% specificity, 77.8% positive predictive value (PPV), and 78.9% negative predictive value (NPV). The most common HPV genotypes detected in FFPE cervical cancer tissue samples were HPV 16 (56%) and HPV 18 (10%). The positivity rate of hTERT and Ki67 mRNA expressions in FFPE cervical cancer tissue samples on RT-qPCR was 65% and 93% respectively. Moreover, the positivity rates were 92% for a combination of HPV E6/E7 and hTERT mRNA expressions, 97% for HPV E6/E7 and Ki67 mRNA expressions, and 99% (99/100) for the combination of HPV E6/E7, hTERT, and Ki67 mRNA expressions. These data showed that SSC FFPE cervical cancer tissue samples correlated more strongly with high Ki67 mRNA expressions than with hTERT mRNA expressions. Notably, hTERT and Ki67 mRNA expression level was increased in high-grade cervical lesions, but was very low in normal samples. Our findings suggest that the combination of HPV E6/E7, hTERT, and Ki67 mRNA expression levels could be used in a complementary manner in diagnosing high-grade cervical lesions. Further studies are required to evaluate these assays as a useful predictive tool for screening low-grade cervical lesions.
Morten, Brianna C.; Scott, Rodney J.; Avery-Kiejda, Kelly A.
p53 is expressed as multiple smaller isoforms whose functions in cancer are not well understood. The p53 isoforms demonstrate abnormal expression in different cancers, suggesting they are important in modulating the function of full-length p53 (FLp53). The quantification of relative mRNA expression has routinely been performed using real-time PCR (qPCR). However, there are serious limitations when detecting p53 isoforms using this method, particularly for formalin-fixed paraffin-embedded (FFPE) tissues. The use of FFPE tumours would be advantageous to correlate expression of p53 isoforms with important clinical features of cancer. One alternative method of RNA detection is the hybridization-based QuantiGene 2.0 Assay, which has been shown to be advantageous for the detection of RNA from FFPE tissues. In this pilot study, we compared the QuantiGene 2.0 Assay to qPCR for the detection of FLp53 and its isoform Δ40p53 in matched fresh frozen (FF) and FFPE breast tumours. FLp53 mRNA expression was detected using qPCR in FF and FFPE tissues, but Δ40p53 mRNA was only detectable in FF tissues. Similar results were obtained for the QuantiGene 2.0 Assay. FLp53 relative mRNA expression was shown to be strongly correlated between the two methods (R2 = 0.9927, p = 0.0031) in FF tissues, however Δ40p53 was not (R2 = 0.4429, p = 0.3345). When comparing the different methods for the detection of FLp53 mRNA from FFPE and FF samples, no correlation (R2 = 0.0002, p = 0.9863) was shown using the QuantiGene 2.0 Assay, and in contrast, the level of expression was highly correlated between the two tissues using qPCR (R2 = 0.8753, p = 0.0644). These results suggest that both the QuantiGene 2.0 Assay and qPCR methods are inadequate for the quantification of Δ40p53 mRNA in FFPE tissues. Therefore, alternative methods of RNA detection and quantification are required to study the relative expression of Δ40p53 in FFPE samples. PMID:27832134
Kriegsmann, Mark; Randau, Thomas M; Gravius, Sascha; Lisenko, Katharina; Altmann, Carolin; Arens, Norbert; Kriegsmann, Jörg
Rheumatoid arthritis (RA) is a chronic autoimmune disease with a heterogeneous clinical presentation affecting about 1 % of adults in developed countries. Currently, the diagnosis is based on the revised criteria of the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) from 2010. These criteria include clinical and laboratory parameters. Because of the variability of the clinical picture, delayed diagnosis of RA occurs in a significant subset of patients. Therefore, the discovery of novel biomarkers that improve the diagnosis of RA is of particular interest. Recently, it became evident that miRNAs have regulatory activities in physiologic processes and human diseases. Upregulation of miR-146a, miR-155, and miR-223 has been shown in various compartments such as serum, blood, synovial fluid, and tissues in patients with RA. A total of 87 samples were analyzed (RA 50, osteoarthritis (OA) 37). RNA was isolated from formalin-fixed paraffin-embedded synovial tissue (FFPE). The relative expression of miR-146a, miR-155, and miR-223 was determined by comparison to a housekeeping RNA molecule (snRNA U6) and an RNA pool from histologically and clinically verified OA samples. miR-146a, miR-155, and miR-223 were significantly elevated in RA compared to OA synovial tissues (p < 0.001). A strong correlation between the miRNAs could be observed. The sensitivity and specificity for the detection of RA were 0.76/0.80 (miR-146a), 0.80/0.95 (miR-155), and 0.86/0.81 (miR-223). The combination of miR-155 and miR-223 resulted in the highest area under the curve (AUC 0.92) with a sensitivity and specificity of 0.84/0.91, respectively. Significantly higher expression levels of miR-146a, miR-155, and miR-223 in FFPE synovial tissue samples of patients with established RA compared to patients with OA were shown. The usefulness of these miRs for the differential diagnosis of early phases of RA against OA remains to be investigated.
Detection of the synovial sarcoma translocation t(X;18) (SYT;SSX) in paraffin-embedded tissues using reverse transcriptase-polymerase chain reaction: a reliable and powerful diagnostic tool for pathologists. A molecular analysis of 221 mesenchymal tumors fixed in different fixatives.
Guillou, L; Coindre, J; Gallagher, G; Terrier, P; Gebhard, S; de Saint Aubain Somerhausen, N; Michels, J; Jundt, G; Vince, D R; Collin, F; Trassard, M; Le Doussal, V; Benhattar, J
Synovial sarcoma (SS) is a relatively rare sarcoma, which may be confused with several other mesenchymal and nonmesenchymal lesions. It bears the t(X;18) (SYT;SSX) translocation, which seems to be specific for this tumor type and can be detected in paraffin-embedded tissue, using reverse transcriptase-polymerase chain reaction (RT-PCR). However, the specificity and sensitivity of this detection method have rarely been examined in a large series. Using RT-PCR, we examined 250 mesenchymal and nonmesenchymal, benign and malignant, paraffin-embedded lesions for the SS t(X;18) (SYT-SSX) translocation. PCR products were obtained from 221 tumors (88.5%). There were 135 non-SS tumors, 22 biphasic, and 64 monophasic spindle/round cell SS, of which 10 were cytogenetically confirmed as t(X;18)-positive. SYT-SSX gene fusion transcripts were detected in the SS tumor category only (100% specificity), including 100% of the biphasic SS and 86% of monophasic spindle/round cell SS. Nine tumors originally diagnosed as SS were t(X;18) (SYT-SSX)-negative. Following reassessment, only 3 of these tumors showed clinicopathologic, immunohistochemical, and/or ultrastructural features consistent with that diagnosis, thus raising the overall detection sensitivity to 96%. With regard to the potential adverse effect of the fixatives used, PCR products were obtained in 100%, 91.5%, 90.5%, and 0% of tumors fixed with AFA, buffered formalin, Holland Bouin, and conventional Bouin's fluid, respectively. This study shows that the detection of the SS t(X;18) (SYT-SSX) in paraffin-embedded tissue is feasible with a 100% specificity and an overall 96% sensitivity, provided non-Bouin's fluid fixation is used.
... the right diagnosis. What should a person do days before a kidney biopsy? Days before the procedure, ... procedure. What can a person expect on the day of the kidney biopsy? A person should arrive ...
Elaba, Zendee; Hughey, Lauren; Isayeva, Tatyana; Weeks, Beth; Solovan, Caius; Solovastru, Laura; Andea, Aleodor
Trichodysplasia spinulosa (TS) is a rare and only recently characterized cutaneous disease occurring in immunocompromised patients. The disease is characterized by spiny follicular papules on clinical examination and by the presence of viral inclusions at ultrastructural examination. In the last year, this virus has been identified as a new member of the polyomavirus family and designated as TS-associated polyomavirus (TSPyV). We report two organ transplant patients with this disease in which we were able to identify the TSPyV at ultrastructural and molecular level from formalin-fixed paraffin-embedded biopsies of lesional skin. Similar to prior described cases, the patients presented with follicular papules which were concentrated on the central face and associated with alopecia. Histopathology of both cases showed dilated follicular infundibula plugged with cornified eosinophilic cells containing large trichohyaline granules. Transmission electron microscopy on paraffin-embedded tissue in case 1 showed 28-nm intracellular viral particles morphologically consistent with polyoma virus. For both cases the presence of TSPyV was confirmed by polymerase chain reaction with virus-specific primers followed by identification by direct sequencing. These two cases show the presence of the newly described TSPyV in TS further establishing its association with this distinctive disease.
... biopsy; Pap smear - cone biopsy; HPV - cone biopsy; Human papilloma virus - cone biopsy; Cervix - cone biopsy; Colposcopy - cone biopsy Images Female reproductive anatomy Cold cone biopsy Cold cone removal References American ...
... for a liver biopsy by talking with a health care provider having blood tests arranging for a ride home fasting before the ... for a liver biopsy by talking with a health care provider having blood tests arranging for a ride home fasting before the ...
Biopsy - gingiva (gums) ... the mouth in the area of the abnormal gum tissue. You may also have an injection of numbing medicine. A small piece of gum tissue is removed and checked for problems in ...
Detection of ASPL/TFE3 fusion transcripts and the TFE3 antigen in formalin-fixed, paraffin-embedded tissue in a series of 18 cases of alveolar soft part sarcoma: useful diagnostic tools in cases with unusual histological features.
Williams, Ann; Bartle, Gillian; Sumathi, Vaiyapuri P; Meis, Jeanne M; Mangham, D Chas; Grimer, Rob J; Kindblom, Lars-Gunnar
Alveolar soft part sarcoma (ASPS) is a rare malignancy; diagnostic problems may occur when cases present as a metastasis or with unusual morphologic features. In this study, a series of 18 cases with follow-up information were analysed with regard to the ASPL/TFE3 fusion transcripts and immuno-detection of TFE3 using archival formalin-fixed, paraffin-embedded tissues. Novel primers to detect ASPL/TFE3 fusion transcripts, type 1 and 2, were designed. The patients, ten female and eight male, ranged in age from 3 to 46 years; 16 involved soft tissues of the extremities (nine, lower; seven, upper), one involved the uterine cervix and one was a primary bone tumour of the foot. Seven ASPS had unusual morphologic features lacking the typical alveolar pattern. Seven had lung metastases at the time of diagnosis, and three developed lung and brain metastases later. Four patients died of disease (after 1-5 years); four are alive with metastases (after 2-15 years), and ten are alive and well (after 1-10 years). Vascular invasion correlated with metastatic disease. All 18 ASPS, four granular cell tumours (one of which was malignant) and one adrenal cortical carcinoma showed TFE3 immuno-positivity. The 18/18 ASPS showed ASPL/TFE3 fusion transcripts (nine, type 1; nine, type 2), four of which had a balanced translocation. ASPL/TFE3 fusion transcripts were not detected in 25 controls. We conclude that immuno-detection of TFE3 and RT-PCR-based identification of ASPL/TFE3 fusion transcripts in formalin-fixed, paraffin-embedded tissues are powerful tools in the diagnosis of ASPS, particularly in cases with unusual morphologic features.
Tholouli, Eleni; Hoyland, Judith A.; Di Vizio, Dolores; O'Connell, Fionnuala; MacDermott, Sarah A.; Twomey, David; Levenson, Richard; Yin, John A. Liu; Golub, Todd R.; Loda, Massimo; Byers, Richard . E-mail: email@example.com
Gene expression mapping using microarray analysis has identified useful gene signatures for predicting outcome. However, little of this has been translated into clinically effective diagnostic tools as microarrays require high quality fresh-frozen tissue samples. We describe a methodology of multiplexed in situ hybridization (ISH) using a novel combination of quantum dot (QD)-labeled oligonucleotide probes and spectral imaging analysis in routinely processed, formalin-fixed paraffin embedded human biopsies. The conditions for QD-ISH were optimized using a poly d(T) oligonucleotide in decalcified bone marrow samples. Single and multiplex QD-ISH was performed in samples with acute leukemia and follicular lymphoma using oligonucleotide probes for myeloperoxidase, bcl-2, survivin, and XIAP. Spectral imaging was used for post hybridization tissue analysis, enabling separation of spatially colocalized signals. The method allows quantitative characterization of multiple gene expression using non-bleaching fluorochromes. This is expected to facilitate multiplex in situ transcript detection in routinely processed human clinical tissue.
... NSAIDs), such as aspirin and ibuprofen Herbal supplements Vitamins Continue to take any prescription medicines unless your provider tells you not to take them. Your provider may ask you to: Eat only light meals the day before the biopsy. Do an ...
Singh, Usha; Singh, Rajeev; Santosh, Deepa; Parkash, Jai; Singh, Rana Gopal; Singh, Shivendra
Introduction Hypothyroidism is prevalent in India. Its association with renal diseases though not very common but have been described in many studies. Here we are reporting renal biopsy findings in 16 cases, all of whom were already diagnosed cases of hypothyroidism. Aim To study renal parenchymal diseases associated in patients with hypothyroidism. Materials and Methods Formalin fixed paraffin embedded sections of renal biopsy were examined after staining with H&E, PAS and Acid Fuschin Orange G (AFOG) stain. Serum urea/creatinine measurements done by semi-autoanalysers and urine analysis were done by using urine strips and light microscopy. Results In 16 cases, M:F ratio was 9:7. Duration of disease varied from 6 months to 14 years. Blood urea and serum creatinine were raised in 10 cases (62.5%) and nephrotic range proteinuria was present in 13 cases (81.25%). Two of the patients had co existing systemic lupus erythaematous. Renal pathology revealed membranous glomerulonephritis (GN) in both cases. In renal biopsy seven cases (43.75%) had pure Membranous Glomerulonephritis (MGN), 4 cases (25%) had mixture of Mesan-gial cell proliferation and membranous Glomerulonephritis(GN) also called MembranoProliferative GN (MPGN). Another four cases (25%) had Focal Segmental Glomerulosclerosis (FSGS) with chronic interstitial nephritis and one case was having minimal change disease. Conclusion Thus present study concludes that hypothyroidism can cause renal parenchymal disease like membranous GN, mesangiocapillary GN which is also called as membranoproliferative GN and FSGS. PMID:27656449
Comparison of targeted next-generation sequencing (NGS) and real-time PCR in the detection of EGFR, KRAS, and BRAF mutations on formalin-fixed, paraffin-embedded tumor material of non-small cell lung carcinoma-superiority of NGS.
Tuononen, Katja; Mäki-Nevala, Satu; Sarhadi, Virinder Kaur; Wirtanen, Aino; Rönty, Mikko; Salmenkivi, Kaisa; Andrews, Jenny M; Telaranta-Keerie, Aino I; Hannula, Sari; Lagström, Sonja; Ellonen, Pekka; Knuuttila, Aija; Knuutila, Sakari
The development of tyrosine kinase inhibitor treatments has made it important to test cancer patients for clinically significant gene mutations that influence the benefit of treatment. Targeted next-generation sequencing (NGS) provides a promising method for diagnostic purposes by enabling the simultaneous detection of multiple mutations in various genes in a single test. The aim of our study was to screen EGFR, KRAS, and BRAF mutations by targeted NGS and commonly used real-time polymerase chain reaction (PCR) methods to evaluate the feasibility of targeted NGS for the detection of the mutations. Furthermore, we aimed to identify potential novel mutations by targeted NGS. We analyzed formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens from 81 non-small cell lung carcinoma patients. We observed a significant concordance (from 96.3 to 100%) of the EGFR, KRAS, and BRAF mutation detection results between targeted NGS and real-time PCR. Moreover, targeted NGS revealed seven nonsynonymous single-nucleotide variations and one insertion-deletion variation in EGFR not detectable by the real-time PCR methods. The potential clinical significance of these variants requires elucidation in future studies. Our results support the use of targeted NGS in the screening of EGFR, KRAS, and BRAF mutations in FFPE tissue material.
De Rossi, Scott S; Ciarrocca, Katharine
Oral lichen planus (OLP) is commonly found in middle-aged women. Although the cause is unknown, research points to several complex immunologic events and cells that are responsible for the inflammatory destruction and chronicity of these lesions. Biopsy for histologic diagnosis is recommended. The mainstay of treatment remains topical corticosteroids; however, newer therapies such as immunomodulating agents are available for recalcitrant lesions. In cases of lichenoid mucositis or reactions, treatment should be directed at identifying and removing the presumed cause. Given the apparent risk of squamous cell carcinoma in these patients, frequent follow-up and repeat biopsy are vital.
Zeuss, M S; Miller, C S; White, D K
Commercial biotinylated DNA probes specific for human papillomavirus (HPV) types 6 and 11; 16 and 18; and 31, 33, and 35 were used for in situ hybridization analysis of 105 oral mucosal specimens from 5 cases of verruca vulgaris, 15 cases of condyloma acuminatum, 30 cases of squamous papilloma, 20 cases of hyperkeratosis/acanthosis, 15 cases of epithelial dysplasia, 5 cases of carcinoma in situ, and 15 cases of squamous cell carcinoma. Positive hybridization signals were found in 26 specimens (24.8%). Only HPV-6/11 was detected. HPV DNA occurred significantly more often (p less than 0.005, chi-square analysis) in condyloma acuminatum (100%) and verruca vulgaris (100%) than squamous papilloma (13.3%), hyperkeratotic/acanthotic lesions (10%), and malignant and premalignant lesions (0%). The tongue (19.1%) and labial epithelium (17.1%) were infected most frequently. Nuclear reaction products indicating HPV infection were associated primarily with koilocytes. These results demonstrate the usefulness of commercial biotinylated probes for HPV DNA analysis in routine paraffin-embedded lesion specimens. They confirm HPV involvement in benign lesions of the oral mucosa but fail to associate HPV infection with oral cancer and precancer.
Bone biopsy; Biopsy - bone ... the cut, then pushed and twisted into the bone. Once the sample is obtained, the needle is ... sample is sent to a lab for examination. Bone biopsy may also be done under general anesthesia ...
A muscle biopsy involves removal of a plug of tissue usually by a needle to be later used for examination. Sometimes ... there is a patchy condition expected an open biopsy may be used. Open biopsy involves a small ...
A bone biopsy is performed by making a small incision into the skin. A biopsy needle retrieves a sample of bone and it ... examination. The most common reasons for bone lesion biopsy are to distinguish between benign and malignant bone ...
Cancer treatment - mucositis; Cancer treatment - mouth pain; Cancer treatment - mouth sores; Chemotherapy - mucositis; Chemotherapy - mouth pain; Chemotherapy - mouth sores; Radiation therapy - mucositis; Radiation therapy - mouth pain; Radiation therapy - mouth ...
Aquaculture species depend more heavily on mucosal barriers than their terrestrial agricultural counterparts as they are continuously interacting with the aquatic microbiota. Unlike classical immune centers, such as the spleen and kidney, the accessibility of mucosal surfaces through immersion/dip t...
Stead, R H; Tomioka, M; Quinonez, G; Simon, G T; Felten, S Y; Bienenstock, J
Inflammatory or allergic conditions, as well as situations where healing and repair processes occur, are characterized by the presence of increased numbers of mast cells. Previous work on the effect of neuropeptides on mast cell mediator release showed that only substance P caused such release from intestinal mucosal mast cells [Shanahan, F., Denburg, J. A., Fox, J., Bienenstock, J. & Befus, A. D. (1985) J. Immunol. 135, 1331-1337]. Accordingly, we investigated the microanatomical relationship between mast cells and enteric nerves in normal rat intestine and parasite-infected rat intestine, in which mucosal mast cell hyperplasia occurs. Combined immunohistochemistry for neuron-specific enolase and staining with alcian blue at pH 0.5 was employed on paraffin-embedded sections of normal and Nippostrongylus brasiliensis-infected rat jejunum. Sixty-seven percent of intestinal mucosal mast cells were touching subepithelial nerves, and an additional 20% were within 2 micron of nerves. Assessment of the proportion of the lamina propria occupied by mast cells (12.5%), the average mast cell area (121 +/- 28 microns 2), and the density of enteric nerves (one per 788 +/- 151 microns 2) suggested that the association was 5 times greater than would be expected by chance alone (P less than 0.0001). In consecutive sections, the nerves in contact with mast cells were also shown to contain substance P and/or calcitonin-gene-related peptide. Electron microscopy confirmed this association: 8% of the mast cells in infected rats exhibited membrane-membrane contact with unmyelinated axons containing 70- to 170-nm dense-core vesicles, and an additional 31% were situated less than 250 nm from nerves. Other mast cells appeared to embrace nerve bundles through the projection of lamellopodia. These data provide systematic quantitative evidence that a structural foundation for communication between the immune and nervous systems exists in the rat gastrointestinal tract. Images PMID:2437589
Amemiya, Takeshi; Nakamura, Takahiro; Yamamoto, Toshiro; Kinoshita, Shigeru; Kanamura, Narisato
The human amniotic membrane (AM) is a thin intrauterine placental membrane that is highly biocompatible and possesses anti-inflammatory and anti-scarring properties. Using AM, we developed a novel method for cultivating oral mucosal epithelial cell sheets. We investigated the autologous transplantation of oral mucosal epithelial cells cultured on AM in patients undergoing oral surgeries. We obtained specimens of AM from women undergoing cesarean sections. This study included five patients without any history of a medical disorder who underwent autologous cultured oral epithelial transplantation following oral surgical procedures. Using oral mucosal biopsy specimens obtained from these patients, we cultured oral epithelial cells on an AM carrier. We transplanted the resultant cell sheets onto the oral mucosal defects. Patients were followed-up for at least 12 months after transplantation. After 2–3 weeks of being cultured on AM, epithelial cells were well differentiated and had stratified into five to seven layers. Immunohistochemistry revealed that the cultured cells expressed highly specific mucosal epithelial cell markers and basement membrane proteins. After the surgical procedures, no infection, bleeding, rejection, or sheet detachment occurred at the reconstructed sites, at which new oral mucous membranes were evident. No recurrence was observed in the long-term follow-up, and the postoperative course was excellent. Our results suggest that AM-cultured oral mucosal epithelial cell sheets represent a useful biomaterial and feasible method for oral mucosal reconstruction. However, our primary clinical study only evaluated their effects on a limited number of small oral mucosal defects. PMID:25915046
PS, Satheesh Kumar; Balan, Anita; Sankar, Arun; Bose, Tinky
Patients receiving radiotherapy or chemotherapy will receive some degree of oral mucositis The incidence of oral mucositis was especially high in patients: (i) With primary tumors in the oral cavity, oropharynx, or nasopharynx; (ii) who also received concomitant chemotherapy; (iii) who received a total dose over 5,000 cGy; and (iv) who were treated with altered fractionation radiation schedules. Radiation-induced oral mucositis affects the quality of life of the patients and the family concerned. The present day management of oral mucositis is mostly palliative and or supportive care. The newer guidelines are suggesting Palifermin, which is the first active mucositis drug as well as Amifostine, for radiation protection and cryotherapy. The current management should focus more on palliative measures, such as pain management, nutritional support, and maintenance, of good oral hygiene PMID:20668585
Orotate phosphoribosyl transferase (OPRT) is the initial enzyme of 5-FU activation, in which 5-FU is converted to 5-fluorouridinemonophosphate. Dihydropyrimidine dehydrogenase (DPD) is a degrading enzyme that catabolizes 5-FU. In this study, we investigated the expression of these enzymes in normal prostate gland (NP), hormone-sensitive prostate cancer (HSPC), and hormone-refractory prostate cancer (HRPC). The prostatic tissue specimens were obtained from patients who had undergone prostate needle biopsies without any treatments or with PSA failure after initial androgen deprivation. The tissue samples derived from formalin-fixed, paraffin-embedded (FFPE) sections were prepared by laser-capture microdissection, and from them RNA was extracted. The levels of OPRT and DPD mRNA expression were examined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The level of OPRT mRNA expression in the HSPC or the HRPC specimens was significantly higher than that in the NP specimens. There was a significant correlation between OPRT mRNA expression levels and the tumor pathological grade. Furthermore, the OPRT/ DPD expression ratio, a powerful predictive factor to evaluate 5-FU sensitivity, in the HRPC group was significantly higher than that in the low-grade HSPC group. Thus, the quantitative evaluation for these enzymes based on phosphorylation of 5-FU may be an effective option for some prostate cancer patients, particularly HRPC group.
Alexandrescu, Doru T; Kauffman, C Lisa; Jatkoe, Timothy A; Hartmann, Dan P; Vener, Tatiana; Wang, Haiying; Derecho, Carlo; Rajpurohit, Yashoda; Wang, Yixin; Palma, John F
Diagnosis of cutaneous melanoma requires accurate differentiation of true malignant tumors from highly atypical lesions, which lack the capacity to develop uncontrolled proliferation and to metastasize. We used melanoma markers from previous work to differentiate benign and atypical lesions from melanoma using paraffin-embedded tissue. This critical step in diagnosis generates the most uncertainty and discrepancy between dermatopathologists. A total of 193 biopsy tissues were selected: 47 melanomas, 48 benign nevi, and 98 atypical/suspicious, including 48 atypical nevi and 50 melanomas as later assigned by expert dermatopathologists. Performance for SILV, GDF15, and L1CAM normalized to TYR in unequivocal melanoma versus benign nevi resulted in an area under the curve (AUC) of 0.94, 0.67, and 0.5, respectively. SILV also differentiated atypical cases classified as melanoma from atypical nevi with an AUC=0.74. Furthermore, SILV showed a significant difference between suspicious melanoma and each suspicious atypia group: melanoma versus severe atypia and melanoma versus moderate atypia had P-values of 0.0077 and 0.0009, respectively. SILV showed clear discrimination between melanoma and benign unequivocal cases as well as between different atypia subgroups in the group of suspicious samples. The role and potential utility of this molecular assay as an adjunct to the morphological diagnosis of melanoma are discussed.
Abstract The mucosal surfaces (skin, gill, and intestine) constitute the first line of defense against pathogen invasion while simultaneously carrying out a diverse array of other critical physiological processes, including nutrient absorption, osmoregulation, and waste excretion. Aquaculture specie...
Konturek, J W; Dembinski, A; Stoll, R; Domschke, W; Konturek, S J
The gastropathy associated with the ingestion of non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin is a common side effect of this class of drugs, but the precise mechanisms by which they cause mucosal damage have not been fully explained. During continued use of an injurious substance, such as aspirin, the extent of gastric mucosal damage decreases and this phenomenon is named gastric adaptation. To assess the extent of mucosal damage by aspirin and subsequent adaptation the effects of 14 days of continuous, oral administration of aspirin (2 g per day) to eight healthy male volunteers was studied. To estimate the rate of mucosal damage, gastroscopy was performed before (day 0) and at days 3, 7, 14 of aspirin treatment. Gastric microbleeding and gastric mucosal blood flow were measured using laser Doppler flowmeter and mucosal biopsy specimens were taken for the estimation of tissue DNA synthesis and RNA and DNA concentration. In addition, the activation of neutrophils in peripheral blood was assessed by measuring their ability to associate with platelets. Aspirin induced acute damage mainly in gastric corpus, reaching at day 3 about 3.5 on the endoscopic Lanza score but lessened to about 1.5 at day 14 pointing to the occurrence of gastric adaptation. Mucosal blood flow increased at day 3 by about 50% in the gastric corpus and by 88% in the antrum. The in vitro DNA synthesis and RNA concentration, an index of mucosal growth, were reduced at day 3 but then increased to reach about 150% of initial value at the end of aspirin treatment. It is concluded that the treatment with aspirin in humans induces gastric adaptation to this agent, which entails the increase in mucosal blood flow, the rise in neutrophil activation, and the enhancement in mucosal growth. PMID:7959223
Bauer, John J.; Zeng, Jianchao; Weir, James; Zhang, Wei; Sesterhenn, Isabell A.; Connelly, Roger R.; Moul, Judd W.; Mun, Seong K.
Urologists routinely use the systematic sextant needle biopsy technique to detect prostate cancer. However, recent evidence suggests that this technique has a significant sampling error. We have developed a novel 3D computer assisted prostate biopsy simulator based upon 201 whole- mounted step-sectioned radical prostatectomy specimens to compare the diagnostic accuracy of various prostate needle biopsy protocols. Computerized prostate models have been developed to accurately depict the anatomy of the prostate and all individual tumor foci. We obtained 18-biopsies of each prostate model to determine the detection rates of various biopsy protocols. As a result, the 10- and 12- pattern biopsy protocols had a 99.0 percent detection rate, while the traditional sextant biopsy protocol rate was only 72.6 percent. The 5-region biopsy protocol had a 90.5 percent detection rate. the lateral sextant pattern revealed a detection rate of 95.5 percent, whereas the 4-pattern lateral biopsy protocol had a 93.5 percent detection rate. Our results suggest that all the biopsy protocols that use laterally placed biopsies based upon the five region anatomical model are superior to the routinely used sextant prostate biopsy pattern. Lateral biopsies in the mid and apical zones of the gland are the most important.
Background Mucosal biopsy is the most common sampling technique used to assess microbial communities associated with the intestinal mucosa. Biopsies disrupt the epithelium and can be associated with complications such as bleeding. Biopsies sample a limited area of the mucosa, which can lead to potential sampling bias. In contrast to the mucosal biopsy, the mucosal brush technique is less invasive and provides greater mucosal coverage, and if it can provide equivalent microbial community data, it would be preferable to mucosal biopsies. Results We compared microbial samples collected from the intestinal mucosa using either a cytology brush or mucosal biopsy forceps. We collected paired samples from patients with ulcerative colitis (UC) who had previously undergone colectomy and ileal pouch anal anastomosis (IPAA), and profiled the microbial communities of the samples by sequencing V4-V6 or V4-V5 16S rRNA-encoding gene amplicons. Comparisons of 177 taxa in 16 brush-biopsy sample pairs had a mean R2 of 0.94. We found no taxa that varied significantly between the brush and biopsy samples after adjusting for multiple comparisons (false discovery rate ≤0.05). We also tested the reproducibility of DNA amplification and sequencing in 25 replicate pairs and found negligible variation (mean R2 = 0.99). A qPCR analysis of the two methods showed that the relative yields of bacterial DNA to human DNA were several-fold higher in the brush samples than in the biopsies. Conclusions Mucosal brushing is preferred to mucosal biopsy for sampling the epithelial-associated microbiota. Although both techniques provide similar assessments of the microbial community composition, the brush sampling method has relatively more bacterial to host DNA, covers a larger surface area, and is less traumatic to the epithelium than the mucosal biopsy. PMID:24529162
Nerve biopsy is the removal of a small piece of nerve for examination. Through a small incision, a sample ... is removed and examined under a microscope. Nerve biopsy may be performed to identify nerve degeneration, identify ...
Sentinel node biopsy is a technique which helps determine if a cancer has spread (metastasized), or is contained locally. When a ... is closest to the cancer site. Sentinel node biopsy is used to stage many kinds of cancer, ...
Iwama, H; Horikoshi, S; Shirato, I; Tomino, Y
To determine the relationship between the detection of Epstein-Barr virus (EBV)-specific DNA and glomerular injury, 33 renal needle-biopsy specimens that had been formalin-fixed and paraffin-embedded were analyzed using polymerase chain reaction (PCR) with subsequent nonradioactive Southern blot technique. Light microscopic examination and immunofluorescence were also performed. In 30 of 33 renal biopsy specimens, the beta globin gene could be successfully amplified as integrity controls. These 30 patients consisted of 12 patients with immunoglobulin A nephropathy (IgAN), 10 patients with minor glomerular abnormalities, 6 patients with membranous nephropathy, and 2 patients with focal/segmental lesions. EBV was detected in 7 of 12 patients with IgAN (58%), 3 of 6 patients with membranous nephropathy (50%), 0 of 10 patients with minor glomerular abnormalities (0%), and 2 of 2 patients with focal/segmental lesions. EBV detection was not disease specific. The EBV detection ratio of the group with glomerular mesangial lesions (64%; 9 of 14 patients) was significantly greater than those without (19%; 3 of 16 patients; P < 0.012, chi-square test). The EBV detection ratio of the group with glomerular lesions (60%; 12 of 20 patients) was significantly greater than those without (0%; 0 of 10 patients; P < 0.0016, Fisher's exact test), and the EBV detection ratio of the group with fibrinogen deposits observed in immunofluorescence (73%; 11 of 15 patients) was significantly greater than those without (7%; 1 of 15 patients; P < 0.0002, chi-square test). The EBV detection ratio of the group with immunoglobulin deposits (57%; 12 of 21 patients) was also significantly greater than those without (0%; 0 of 9 patients; P < 0.0040, Fisher's exact test). These data suggest that EBV can damage the glomerular mesangium beyond disease units and be mediated by immunoglobulin in patients with various chronic glomerulonephritides.
Zhang, Xuchen; Ouyang, Jie; Wieczorek, Rosemary; DeSoto, Fidelina
Severe gastrointestinal erosion, ulcer, necrosis and strictures after an acute iron overdose are well described. However, gastric mucosal injury in patients receiving therapeutic iron has received only scant recognition despite its wide use. We report a case of iron medication-induced gastric mucosal injury in a 76-year-old male who presented with iron deficiency anemia and had been taking ferrous sulfate tablet for 4 years. Esophagogastroduodenoscopy (EGD) revealed a pale, villous appearing flat lesion along the lesser curvature of gastric body. Histopathologic examination of EGD biopsies of the flat lesion showed brown crystalline materials deposited in the lamina propria of gastric mucosa, which was accompanied with fibrosis, chronic inflammation, and foreign body reaction. The crystalline materials were covered and admixed with gastric epithelium. Prussian blue iron stain confirmed that the brown crystalline materials were iron. The iron and hemosiderin accumulation was also seen in cytoplasm of epithelial cells and lumen of fundic gastric glands. The recognition and reporting by pathologists of iron-induced changes in EGD biopsies will alert clinicians to this underrecognized but easily correctable complication by alternative forms of iron therapy, such as liquid preparation.
Blum, Andreas; Beck-Zoul, Ulrike; Held, Laura; Haase, Sylvie
Background Hypomelanotic or amelanotic melanomas are challenging to identify, especially at mucosal sites. The dermoscopic clues to the diagnosis of mucosal melanomas have been reported to be structureless zones with the presence of blue, gray, or white colors. Case A female in her seventies noted a new lesion on the inside of her right labia that first appeared two months prior. Her past medical history was significant for rheumatoid arthritis requiring ongoing treatment with methotrexate for 20 years and adalimumab for 10 years. After no response to two weeks of local treatment for suspected herpes simplex infection, her gynecologist performed a skin biopsy. Based on the histopathological diagnosis of an amelanotic melanoma (Breslow thickness of 1.3 mm) the patient was referred to dermatology for further assessment. Polarized dermoscopy revealed a distinct asymmetric, sharply demarcated homogenous white papule (4 × 5 mm) as well as polymorphous vessels. Conclusion Dermoscopy may aid in the diagnosis of amelanotic mucosal melanomas. Our case revealed a structureless white area and polymorphous vessels. Additional clues to the diagnosis were the advanced age of the patient and the clinical presentation of a new lesion. PMID:27867742
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
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
Anastasiadis, Anastasios; Zapała, Lukasz; Cordeiro, Ernesto; Antoniewicz, Artur; Dimitriadis, Georgios; De Reijke, Theo
Biopsy of the prostate is a common procedure with minor complications that are usually self-limited. However, if one considers that millions of men undergo biopsy worldwide, one realizes that although complication rate is low, the number of patients suffering from biopsy complications should not be underestimated and can be a clinically relevant problem for healthcare professionals. In this review, the authors present diagnosis and management of postbiopsy of prostate complications. Bleeding is the most common complication observed after prostate biopsy, but the use of aspirin or nonsteroidal anti-inflammatory drugs is not an absolute contraindication to prostate biopsy. Emerging resistance to ciprofloxacin is the most probable cause of the increasing risk of infectious complications after prostate biopsy. Even though extremely rare, fatal complications are possible and were described in case reports.
Goodrich, J A; Difiore, R J; Tippens, J K
The orthopedic surgeon is frequently confronted with the decision of when to perform a bone biopsy and whether to do a needle biopsy or an open biopsy. Frequently consultations are received from other services requesting bone biopsies with questionable indications. The indications and contraindications for performing bone biopsies are discussed as well as advantages and disadvantages of either closed or open technique. Four selective cases are discussed with illustrations. The challenge of undiagnosed osseous lesions is best met by rational evaluation of each individual case and coordinated with the team effort of the primary care physician, surgeon, pathologist, and radiologist. The decision for either an open or closed biopsy technique must be based on the experience and skills of the surgeon and pathologist.
Afzali, B; Chapman, E; Racapé, M; Adam, B; Bruneval, P; Gil, F; Kim, D; Hidalgo, L; Campbell, P; Sis, B; Duong Van Huyen, J P; Mengel, M
Precise diagnosis of antibody-mediated rejection (AMR) in cardiac allograft endomyocardial biopsies (EMBs) remains challenging. This study assessed molecular diagnostics in human EMBs with AMR. A set of 34 endothelial, natural killer cell and inflammatory genes was quantified in 106 formalin-fixed, paraffin-embedded EMBs classified according to 2013 International Society for Heart and Lung Transplantation (ISHLT) criteria. The gene set expression was compared between ISHLT diagnoses and correlated with donor-specific antibody (DSA), endothelial injury by electron microscopy (EM) and prognosis. Findings were validated in an independent set of 57 EMBs. In the training set (n = 106), AMR cases (n = 70) showed higher gene set expression than acute cellular rejection (ACR; n = 21, p < 0.001) and controls (n = 15, p < 0.0001). Anti-HLA DSA positivity was associated with higher gene set expression (p = 0.01). Endothelial injury by electron microscopy strongly correlated with gene set expression, specifically in AMR cases (r = 0.62, p = 0.002). Receiver operating characteristic curve analysis for diagnosing AMR showed greater accuracy with gene set expression (area under the curve [AUC] = 79.88) than with DSA (AUC = 70.47) and C4d (AUC = 70.71). In AMR patients (n = 17) with sequential biopsies, increasing gene set expression was associated with inferior prognosis (p = 0.034). These findings were confirmed in the validation set. In conclusion, biopsy-based molecular assessment of antibody-mediated microcirculation injury has the potential to improve diagnosis of AMR in human cardiac transplants.
de Bazelaire, C; Coffin, A; Cohen, S; Scemama, A; de Kerviler, E
Imaging-guided percutaneous biopsies in patients in oncology provide an accurate diagnosis of malignant tumors. Percutaneous biopsy results are improved by correct use of sampling procedures. The risks of percutaneous biopsy are low and its complications are generally moderate. These risks can be reduced using aids such as blund tip introducers, hydrodissection and correct patient positioning. The multidisciplinary team meetings dialogue between oncologist, surgeon and radiologist correctly defines the indications in order to improve the treatment strategies.
Synovial biopsies, gained either by blind needle biopsy or minimally invasive arthroscopy, offer additional information in certain clinical situations where routine assessment has not permitted a certain diagnosis. In research settings, synovial histology and modern applications of molecular biology increase our insight into pathogenesis and enable responses to treatment with new therapeutic agents to be assessed directly at the pathophysiological level. This review focuses on the diagnostic usefulness of synovial biopsies in the light of actual developments. PMID:19951395
... squamous cells - colposcopy; Pap smear - colposcopy; HPV - colposcopy; Human papilloma virus - colposcopy; Cervix - colposcopy; Colposcopy Images Female reproductive anatomy Colposcopy-directed biopsy Uterus References American College of ...
Aerssens, Jeroen; Camilleri, Michael; Talloen, Willem; Thielemans, Leen; Göhlmann, Hinrich W. H.; Wyngaert, Ilse Van den; Thielemans, Theo; de Hoogt, Ronald; Andrews, Christopher N.; Bharucha, Adil E.; Carlson, Paula J.; Busciglio, Irene; Burton, Duane D.; Smyrk, Thomas; Urrutia, Raul; Coulie, Bernard
BACKGROUND & AIMS Irritable bowel syndrome (IBS) has been associated with mucosal dysfunction,, mild inflammation, and altered colonic bacteria. We used microarray expression profiling of sigmoid colon mucosa to assess whether there are stably expressed sets of genes that suggest there are objective molecular biomarkers associated with IBS. METHODS Gene expression profiling was performed using Affymetrix GeneChips with RNA from sigmoid colon mucosal biopsies from 36 IBS patients and 25 healthy control subjects. RTQ-PCR was used to confirm the data in 12 genes of interest. Statistical methods for microarray data were applied to search for differentially expressed genes, and to assess the stability of molecular signatures in IBS patients. RESULTS Mucosal gene expression profiles were consistent across different sites within the sigmoid colon and were stable on repeat biopsy over ~3 months. Differentially expressed genes suggest functional alterations of several components of the host mucosal immune response to microbial pathogens. The most strikingly increased expression involved a yet uncharacterized gene, DKFZP564O0823. Identified specific genes suggest the hypothesis that molecular signatures may enable distinction of a subset of IBS patients from healthy controls. Using 75% of the biopsies as a validation set to develop a gene profile, the test set (25%) was correctly predicted with ~70% accuracy. CONCLUSIONS Mucosal gene expression analysis shows there are relatively stable alterations in colonic mucosal immunity in IBS. These molecular alterations provide the basis to test the hypothesis that objective biomarkers may be identified in IBS and enhance understanding of the disease. PMID:18237869
Wang, Ai-Hua; Li, Ming; Li, Chang-Qing; Kou, Guan-Jun; Zuo, Xiu-Li; Li, Yan-Qing
The human gut microbiota plays a pivotal role in the maintenance of health, but how the microbiota interacts with the host at the colorectal mucosa is poorly understood. We proposed that confocal laser endomicroscopy (CLE) might help to untangle this relationship by providing in vivo physiological information of the mucosa. We used CLE to evaluate the in vivo physiology of human colorectal mucosa, and the mucosal microbiota was quantified using 16 s rDNA pyrosequencing. The human mucosal microbiota agglomerated to three major clusters dominated by Prevotella, Bacteroides and Lactococcus. The mucosal microbiota clusters did not significantly correlate with the disease status or biopsy sites but closely correlated with the mucosal niche physiology, which was non-invasively revealed by CLE. Inflammation tilted two subnetworks within the mucosal microbiota. Infiltration of inflammatory cells significantly correlated with multiple components in the predicted metagenome, such as the VirD2 component of the type IV secretory pathway. Our data suggest that a close correlation exists between the mucosal microbiota and the colorectal mucosal physiology, and CLE is a clinically available tool that can be used to facilitate the study of the in vivo correlation between colorectal mucosal physiology and the mucosal microbiota. PMID:26916597
Court, Colin M.; Kim, Stephen; Braxton, David R.; Hou, Shuang; Muthusamy, V. Raman; Watson, Rabindra R.; Sedarat, Alireza; Tseng, Hsian-Rong; Tomlinson, James S.
Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare
Kwack, Won G; Ho, Won J; Kim, Jae H; Lee, Jin H; Kim, Eo J; Kang, Hyoun W; Lee, Jun K
Although there are general guidelines on endoscopic biopsy for diagnosing gastric neoplasms, they are predominantly based on outdated literature obtained with fiberscopes without analyses specific to tumor characteristics.This study aims to comprehensively characterize the contemporary endoscopic biopsy by determining the diagnostic yield across different lesion morphologies and histological stages, especially exploring how the number and site of biopsy may influence the overall yield.Biopsy samples from suspected gastric neoplasms were collected prospectively from May 2011 to August 2014 in a tertiary care medical center. A standardized methodology was used to obtain a total of 6 specimens from 2 defined sites per lesion. Rate of positive diagnosis based on the biopsy number and site was assessed for specific gastric lesion morphologies and histological stages.A total of 1080 biopsies from 180 pathologically diagnosed neoplastic lesions in 176 patients were obtained during the study. For depressed/ulcerative and polypoid lesions, the yield was already >99% by the fourth biopsy without further gain from additional biopsies. Lower overall yield was observed for infiltrative lesions (57.1% from 4 biopsies). The site of biopsy did not influence the diagnostic yield except for with infiltrative lesions in which biopsies from thickened mucosal folds were of higher yield than erosive regions.Obtaining 4 specimens may be sufficient for accurate diagnosis of a depressed/ulcerative or polypoid gastric lesion regardless of its histological stage. For infiltrative lesions, at least 5 to 6 biopsies per lesion with more representative sampling from thickened mucosal folds may be preferable.
Biopsy - salivary gland ... You have several pairs of salivary glands that drain into your mouth: A major pair in front of the ears (parotid glands) Another major pair beneath your jaw (submandibular ...
... Performed The test is used to diagnose cancer, sarcoidosis, or an infection (such as tuberculosis): When you ... of lymph nodes and other organs and tissues ( sarcoidosis ) Risks Lymph node biopsy may result in any ...
... of different conditions, such as: Rheumatoid lung disease Sarcoidosis Wegener granulomatosis Risks There is a possibility of ... biopsy Malignant mesothelioma Pulmonary tuberculosis Rheumatoid lung disease Sarcoidosis Simple pulmonary eosinophilia Solitary fibrous tumor Viral pneumonia ...
... of the pleural membrane. Pleural biopsy can diagnose tuberculosis , cancer, and other diseases. If this type of ... lung cancer , malignant mesothelioma , and metastatic pleural tumor ), tuberculosis, other infections, or collagen vascular disease. Risks There ...
... due to a virus, fungus, or parasite Mesothelioma Tuberculosis Risks There is a slight chance of: Air ... More Metastatic pleural tumor Pleural needle biopsy Pulmonary tuberculosis Tumor Review Date 11/4/2014 Updated by: ...
... This procedure is also done for certain infections (tuberculosis, sarcoidosis) and autoimmune disorders . Normal Results Biopsies of ... of disease from one body part to another Tuberculosis Risks There is a risk of puncturing the ...
Matsusaka, Keisuke; Ishikawa, Shumpei; Nakayama, Atsuhito; Ushiku, Tetsuo; Nishimoto, Aiko; Urabe, Masayuki; Kaneko, Nobuyuki; Kunita, Akiko; Kaneda, Atsushi; Aburatani, Hiroyuki; Fujishiro, Mitsuhiro; Seto, Yasuyuki; Fukayama, Masashi
Evaluating HER2 gene amplification is an essential component of therapeutic decision-making for advanced or metastatic gastric cancer. A simple method that is applicable to small, formalin-fixed, paraffin-embedded biopsy specimens is desirable as an adjunct to or as a substitute for currently used HER2 immunohistochemistry and in situ hybridization protocols. In this study, we developed a microfluidics-based digital PCR method for determining HER2 and chromosome 17 centromere (CEP17) copy numbers and estimating tumor content ratio (TCR). The HER2/CEP17 ratio is determined by three variables—TCR and absolute copy numbers of HER2 and CEP17—by examining tumor cells; only the ratio of the latter two can be obtained by digital PCR using the whole specimen without purifying tumor cells. TCR was determined by semi-automatic image analysis. We developed a Tumor Content chart, which is a plane of rectangular coordinates consisting of HER2/CEP17 digital PCR data and TCR that delineates amplified, non-amplified, and equivocal areas. By applying this method, 44 clinical gastric cancer biopsy samples were classified as amplified (n = 13), non-amplified (n = 25), or equivocal (n = 6). By comparison, 11 samples were positive, 11 were negative, and 22 were equivocally immunohistochemistry. Thus, our novel method reduced the number of equivocal samples from 22 to 6, thereby obviating the need for confirmation by fluorescence or dual-probe in situ hybridization to < 30% of cases. Tumor content chart-assisted digital PCR analysis is also applicable to multiple sites in surgically resected tissues. These results indicate that this analysis is a useful alternative to HER2 immunohistochemistry in gastric cancers that can serve as a basis for the automated evaluation of HER2 status. PMID:27119558
Newberry, Rodney D; Lorenz, Robin G
Gastrointestinal associated lymphoid tissue can be divided into loosely organized effector sites, which include the lamina propria and intraepithelial lymphocytes, and more organized structures, such as mesenteric lymph nodes (LNs), Peyer's patches (PPs), isolated lymphoid follicles, and cryptopatches (CPs). These organized structures in the gastrointestinal tract have been hypothesized to play the role of primary lymphoid organ, supporting the extrathymic development of T lymphocytes (CPs), secondary lymphoid organs involved in the induction of the mucosal immune response (PPs), and tertiary lymphoid structures whose function is still under debate (isolated lymphoid follicles). The most widely studied lymphoid structure found in the small intestine is the PP. PPs are secondary lymphoid structures, and their development and function have been extensively investigated. However, single lymphoid aggregates resembling PPs have been also described in humans and in the murine small intestines. These isolated lymphoid follicles have both germinal centers and an overlying follicle-associated epithelium, suggesting that they also can function as inductive sites for the mucosal immune response. This review compares and contrasts the development and function of the four main organized gastrointestinal lymphoid tissues: CPs, isolated lymphoid follicles, PPs, and mesenteric LNs.
Immunohistochemical detection of a novel 22- to 25-kilodalton glycoprotein of Paracoccidioides brasiliensis in biopsy material and partial characterization by using species-specific monoclonal antibodies.
Figueroa, J I; Hamilton, A; Allen, M; Hay, R
Two murine monoclonal antibodies (MAbs) specific to Paracoccidioides brasiliensis (as determined by enzyme-linked immunosorbent assay [ELISA] and Western blot [immunoblot]) were produced by using a modification of standard hybridization protocols, with cyclophosphamide included as an immunomodulator to abolish responses to highly cross-reactive immunodominant epitopes. MAbs PS14 and PS15 are two different clones which exhibit similar characteristics by ELISA and Western blot. They are directed against a 22- to 25-kDa antigen which is present in P. brasiliensis and which could not be identified in other dimorphic fungi by ELISA or Western blot. Partial purification of the antigen was accomplished by isoelectric focusing, and deglycosylation studies suggested that the 22- to 25-kDa antigen is a glycoprotein with a pI of between 4.5 and 5 and that O-linked sugars may be part of the recognized epitope. The MAbs stained the cytoplasm of P. brasiliensis yeast and hyphal cells in cryostat sections of fresh cultures of the fungus. In addition, the MAbs stained the wall of paracoccidioidomycotic granulomas, as well as the cytoplasm of the fungus, as determined by the use of immunofluorescence, immunoperoxidase, and immuno-alkaline phosphatase staining techniques in paraffin-embedded sections of human biopsy material, and they failed to stain granulomas resulting from other clinical conditions. These findings suggest that these MAbs have potential use in the immunohistochemical identification of P. brasiliensis. Images PMID:8077405
Ganguly, Shantanu; Mitchell, Aaron P
Biofilms are microbial communities that form on surfaces and are embedded in an extracellular matrix. C. albicans forms pathogenic mucosal biofilms that are evoked by changes in host immunity or mucosal ecology. Mucosal surfaces are inhabited by many microbial species; hence these biofilms are polymicrobial. Several recent studies have applied paradigms of biofilm analysis to study mucosal C. albicans infections. These studies reveal that the Bcr1 transcription factor is a master regulator of C. albicans biofilm formation under diverse conditions, though the most relevant Bcr1 target genes can vary with the biofilm niche. An important determinant of mucosal biofilm formation is the interaction with host defenses. Finally, studies of interactions between bacterial species and C. albicans provide insight into the communication mechanisms that endow polymicrobial biofilms with unique properties.
Charge Coupled Devices (CCDs) are high technology silicon chips that connect light directly into electronic or digital images, which can be manipulated or enhanced by computers. When Goddard Space Flight Center (GSFC) scientists realized that existing CCD technology could not meet scientific requirements for the Hubble Space Telescope Imagining Spectrograph, GSFC contracted with Scientific Imaging Technologies, Inc. (SITe) to develop an advanced CCD. SITe then applied many of the NASA-driven enhancements to the manufacture of CCDs for digital mammography. The resulting device images breast tissue more clearly and efficiently. The LORAD Stereo Guide Breast Biopsy system incorporates SITe's CCD as part of a digital camera system that is replacing surgical biopsy in many cases. Known as stereotactic needle biopsy, it is performed under local anesthesia with a needle and saves women time, pain, scarring, radiation exposure and money.
The thyroid is a gland located in the neck. It is a part of the endocrine (hormone) system, and plays a major role in regulating ... sample of cells is needed from the thyroid gland a fine needle biopsy can be performed. During ...
Background and aims: Folate has been implicated as a potential aetiological factor for colorectal cancer. Previous research has not adequately exploited concentrations of folate in normal colonic mucosal biopsies to examine the issue. Methods: Logistic regression models were used to estimate ORs ...
Ban, Shinichi; Suzuki, Syunji; Kubota, Kenji; Ohshima, Susumu; Satoh, Hideaki; Imada, Hiroki; Ueda, Yoshihiko
Lanthanum carbonate is a popular chemical which is administered for patients with end-stage kidney disease to reduce the absorption of phosphate, and lanthanum deposition in the gastroduodenal mucosa has recently been reported. The aim of this study was to assess whether any histologic changes of the gastric mucosa are related to the deposition of lanthanum. Twenty-four patients who revealed the histology of lanthanum deposition on gastroduodenal biopsy between 2011 and 2014 were included in the study, and their clinical records and gastroduodenal biopsies obtained from 2011 to 2015 were reviewed, adding the review of gastroduodenal biopsies before 2011 if possible. Analysis of the deposited materials by scanning electron microscopy-energy dispersive x-ray spectroscopy was performed for a representative gastric biopsy. All patients were diagnosed as having renal insufficiency due to chronic kidney disease and treated with dialysis for more than 5 years, with confirmation of lanthanum carbonate use for 22 patients. Of 121 gastric biopsies and 10 duodenal ones between 2011 and 2015, 86 gastric biopsies (71.1%) and 3 duodenal biopsies (30%), respectively, revealed histology consistent with lanthanum deposition, which was confirmed by scanning electron microscopy-energy dispersive x-ray spectroscopy analysis for a representative case. The deposition tended to occur in the gastric mucosa with regenerative change, intestinal metaplasia, or foveolar hyperplasia (P<.05). Such mucosal changes were observed in about half of the gastric biopsy samples obtained prior to 2010, in which no lanthanum deposition was identified irrespective of the gastric mucosal status. Although direct association between lanthanum deposition and clinical symptoms is not clear, the evaluation of the gastric mucosal status (prior to administration) seems to be important to predict lanthanum deposition when lanthanum carbonate is administered for patients with chronic kidney disease treated with
Reeves, R Keith; Evans, Tristan I; Gillis, Jacqueline; Wong, Fay E; Connole, Michelle; Carville, Angela; Johnson, R Paul
Since the vast majority of infections occur at mucosal surfaces, accurate characterization of mucosal immune cells is critically important for understanding transmission and control of infectious diseases. Standard flow cytometric analysis of cells obtained from mucosal tissues can provide valuable information on the phenotype of mucosal leukocytes and their relative abundance, but does not provide absolute cell counts of mucosal cell populations. We developed a bead-based flow cytometry assay to determine the absolute numbers of multiple mononuclear cell types in colorectal biopsies of rhesus macaques. Using 10-color flow cytometry panels and pan-fluorescent beads, cells were enumerated in biopsy specimens by adding a constant ratio of beads per mg of tissue and then calculating cell numbers/mg of tissue based on cell-to-bead ratios determined at the time of sample acquisition. Testing in duplicate specimens showed the assay to be highly reproducible (Spearman R=0.9476, P<0.0001). Using this assay, we report enumeration of total CD45(+) leukocytes, CD4(+) and CD8(+) T cells, B cells, NK cells, CD14(+) monocytes, and myeloid and plasmacytoid dendritic cells in colorectal biopsies, with cell numbers in normal rhesus macaques varying from medians of 4486 cells/mg (T cells) to 3 cells/mg (plasmacytoid dendritic cells). This assay represents a significant advancement in rapid, accurate quantification of mononuclear cell populations in mucosal tissues and could be applied to provide absolute counts of a variety of different cell populations in diverse tissues.
Ram-Wolff, Caroline; Martin-Garcia, Nadine; Bensussan, Armand; Bagot, Martine; Ortonne, Nicolas
, few morphological and phenotypical parameters are helpful in making a differential diagnosis between erythrodermic CTCL and EID using paraffin embedded skin biopsies.
Carmack, Susanne; Taddei, Tamar; Robert, Marie E; Mistry, Pramod; Jain, Dhanpat
Mixed cryoglobulinemia (MC) has a strong association with hepatitis C virus (HCV) infection and is associated with a higher degree of fibrosis and poor response to therapy. Currently, there are no known histological findings on liver biopsy that correlate with the presence of MC in HCV-infected patients, although we have occasionally noted prominent sinusoidal lymphocytosis in MC patients. The goal of this study is to determine whether sinusoidal lymphocytosis is a histological marker of MC in liver biopsies from patients with chronic hepatitis C. The liver clinic database at our institution was searched for chronic hepatitis C patients with MC who underwent liver biopsy during 1999-2005. Ten such cases were identified and were included in the study. Ten chronic hepatitis C MC-negative cases were matched for age and stage of fibrosis to serve as controls. Histological features (sinusoidal lymphocytes, inflammatory activity, acidophil bodies, and fibrosis stage) were evaluated in each biopsy. Clinical and laboratory data (serum protein electrophoresis, liver enzymes, hepatitis C viral load, treatment status, comorbidities, etc.) were also recorded. Formalin-fixed, paraffin-embedded sections were submitted for immunohistochemical analysis using antibodies against CD3, CD20, CD4, CD8, and CD68. Sinusoidal lymphocytes were counted in 5 hpf (40x) on hematoxylin and eosin (H&E) stain, and on CD3 and CD20 immunostains. The number of CD68+ Kupffer cells was also counted in a similar fashion. In the MC-positive versus MC-negative cases, mean fibrosis stage (2.4 vs. 2.4), inflammatory grade (1.7 vs. 2.1), lymphocyte count (359 vs. 128/5 hpf), and Kupffer cell count (239 vs. 220/5 HPF) were assessed. There was a significant increase in sinusoidal T-cell lymphocytes (P < 0.05) in MC-positive cases as compared to MC-negative cases. Nearly all sinusoidal lymphocytes were CD8-positive cells in both groups. Other histological parameters did not differ in the two groups. MC
Said, Hyder; Kaji, Izumi; Kaunitz, Jonathan D.
Purpose of Review To highlight recent developments in the field of gastroduodenal mucosal defense with emphasis on lumen-gut interactions. Recent Findings There has been a growing interest in the physiological functions of luminal chemosensors present from tongue to colon that detect organic molecules in the luminal content associated with nutrient ingestion, usually associated with specialized cells, in particular the enteroendocrine cells. These receptors transduce the release of peptide hormones, in particular proglucagon-derived products such as the glucagon-like-peptides (GLPs), which have profound effects on gut function and on metabolism. Luminal chemosensors transduce GLP release in response to changes in the cellular environment, as part of the mechanism of nutrient chemosensing. GLP-2 has important trophic effects on the intestinal mucosa, including increasing the proliferation rate of stem cells and reducing transmucosal permeability to ions and small molecules, in addition to increasing the rate of duodenal bicarbonate secretion. GLP-1, although traditionally considered an incretin that enhances the effect of insulin on peripheral tissues, also has trophic effects on the intestinal epithelium. Summary A better understanding of the mechanisms that mediate GLP release can further illuminate the importance of nutrient chemosensing as an important component of the mechanism that mediates the trophic effects of luminal nutrients. GLP-1 and -2 are already in clinical use for the treatment of diabetes and intestinal failure. Improved understanding of the control of their release and their end-organ effects will identify new clinical indications and interventions that enhance their release. PMID:26376476
Scully, Crispian; Mignogna, Michele
Pemphigus defines a group of rare mucocutaneous autoimmune diseases of which pemphigus vulgaris (PV) is the most common. The aetiology and pathogenesis of PV are not completely clear, but there is a fairly strong genetic background: ethnic groups such as Ashkenazi Jews and people of Mediterranean and Indian origin are particularly susceptible and there is a link to HLA class II alleles. The initiating event in PV is not clear, but circulating IgG autoantibodies develop, directed particularly against the intercellular cadherin desmoglein 3 (Dsg3) in desmosomes of stratified squamous epithelium. Oral lesions often herald the disease and are initially vesiculobullous, but they rupture readily to leave ulcers. Involvement of other mucosa and skin is almost inevitable and PV is potentially life threatening. The diagnosis is confirmed by biopsy with histological examination and immunostaining. Management is largely by systemic immunosuppression with corticosteroids, usually azathioprine or other agents, but newer treatments with potentially fewer adverse effects look promising.
... Breast Biopsy An ultrasound-guided breast biopsy uses sound waves to help locate a lump or abnormality ... exam. The transducer sends out inaudible, high—frequency sound waves into the body and then listens for ...
Weijenborg, Pim W; Smout, André J P M; Verseijden, Caroline; van Veen, Henk A; Verheij, Joanne; de Jonge, Wouter J; Bredenoord, Albert J
Increased esophageal sensitivity and impaired mucosal integrity have both been described in patients with gastroesophageal reflux disease, but the relationship between hypersensitivity and mucosal integrity is unclear. The aim of the present study was to investigate acid sensitivity in patients with erosive and nonerosive reflux disease and control subjects to determine the relation with functional esophageal mucosal integrity changes as well as to investigate cellular mechanisms of impaired mucosal integrity in these patients. In this prospective experimental study, 12 patients with nonerosive reflux disease, 12 patients with esophagitis grade A or B, and 11 healthy control subjects underwent an acid perfusion test and upper endoscopy. Mucosal integrity was measured during endoscopy by electrical tissue impedance spectroscopy and biopsy specimens were analyzed in Ussing chambers for transepithelial electrical resistance, transepithelial permeability and gene expression of tight junction proteins and filaggrin. Patients with nonerosive reflux disease and esophagitis were more sensitive to acid perfusion compared with control subjects, having a shorter time to perception of heartburn and higher perceived intensity of heartburn. In reflux patients, enhanced acid sensitivity was associated with impairment of in vivo and vitro esophageal mucosal integrity. Mucosal integrity was significantly impaired in patients with esophagitis, displaying higher transepithelial permeability and lower extracellular impedance. Although no significant differences in the expression of tight junction proteins were found in biopsies among patient groups, mucosal integrity parameters in reflux patients correlated negatively with the expression of filaggrin. In conclusion, sensitivity to acid is enhanced in patients with gastroesophageal reflux disease, irrespective of the presence of erosions, and is associated with impaired esophageal mucosal integrity. Mucosal integrity of the esophagus
... Your 1- to 2-Year-Old Aspiration and Biopsy: Bone Marrow KidsHealth > For Parents > Aspiration and Biopsy: Bone Marrow A A A What's in this ... ósea What It Is Bone marrow aspirations and biopsies are performed to examine bone marrow, the spongy ...
... A Week of Healthy Breakfasts Shyness Aspiration and Biopsy: Bone Marrow KidsHealth > For Teens > Aspiration and Biopsy: Bone Marrow A A A What's in this ... Questions What It Is Bone marrow aspirations and biopsies are performed to examine bone marrow, the spongy ...
Rogers, Tova; Pulitzer, Melissa; Marino, Maria L.; Marghoob, Ashfaq A.; Zivanovic, Oliver; Marchetti, Michael A.
Background There are limited studies on the dermoscopic features of mucosal melanoma, particularly early-stage lesions. Described criteria include the presence of blue, gray, or white colors, with a reported sensitivity of 100%. It is unclear if these features will aid in the detection of early mucosal melanoma or improve diagnostic accuracy compared to naked-eye examination alone. Case An Asian female in her fifties was referred for evaluation of an asymptomatic, irregularly pigmented patch of the clitoral hood and labia minora of unknown duration. Her past medical history was notable for Stage IV non-small cell lung cancer. She denied a personal or family history of skin cancer. Dermoscopic evaluation of the vulvar lesion revealed heterogeneous brown and black pigmentation mostly composed of thick lines. There were no other colors or structures present. As the differential diagnosis included vulvar melanosis and mucosal melanoma, the patient was recommended to undergo biopsy, which was delayed due to complications from her underlying lung cancer. Repeat dermoscopic imaging performed three months later revealed significant changes concerning for melanoma, including increase in size, asymmetric darkening, and the appearance of structureless areas and central blue and pink colors. Histopathological examination of a biopsy and subsequent resection confirmed the diagnosis of melanoma in situ. Conclusion Previously described dermoscopic features for mucosal melanoma may not have high sensitivity for early melanomas. Additional studies are needed to define the dermoscopic characteristics of mucosal melanomas that aid in early detection. Health care providers should have a low threshold for biopsy of mucosal lesions that show any clinical or dermoscopic features of melanoma, especially in older women. PMID:27867747
Li, Tao; Zheng, Yuanting; Sun, Hong; Zhuang, Rongyuan; Liu, Jing; Liu, Tianshu; Cai, Weimin
K-Ras gene mutations have been found in most pancreatic cancers; however, conflicting data on the prognostic value of K-Ras mutations in pancreatic cancer have been published. We conducted a meta-analysis to assess its prognostic significance. Literature searches of PubMed, EMBASE, Cochrane Library, Web of Science and Google Scholar were performed through December 2015 to identify publications exploring the association of K-Ras mutation with overall survival. Forty eligible studies involving 3427 patients with pancreatic cancer were included in the present meta-analysis. Our analysis showed a hazard ratio (HR) of negative association with survival of 1.61 [95 % confidence interval (CI) 1.36-1.90; p < 0.01] in K-Ras mutant pancreatic cancer patients. In subgroup analyses, K-Ras mutations detected in tumor tissues and in liquid biopsies had HRs of 1.37 (95 % CI 1.20-1.57; p < 0.01) and 3.16 (95 % CI 2.1-4.71; p < 0.01), respectively. In addition, the HR was higher when K-Ras mutations were detected in fresh frozen samples (HR = 2.01, 95 % CI 1.28-3.16, p = 0.002) than in formalin-fixed, paraffin-embedded (FFPE) samples (HR = 1.29, 95 % CI 1.12-1.49, p < 0.01). Though K-Ras alterations are more frequent among non-East Asian individuals than East Asian individuals, there were no significant differences in HRs of survival between the two ethnic subgroups. In conclusion, this meta-analysis suggests that K-Ras mutations are associated with a worse overall survival in pancreatic cancer patients, especially when mutations are detected in liquid biopsies or fresh frozen tumor tissue samples.
Purpose of review HIV-1 mucosal transmission plays a critical role in HIV-1 infection and AIDS pathogenesis. This review summarizes the latest advances in biological studies of HIV-1 mucosal transmission, highlighting the implications of these studies in the development of microbicides to prevent HIV-1 transmission. Recent findings New studies of initial HIV-1 infection using improved culture models updated the current view of mucosal transmission. Mechanistic studies enhanced our understanding of cell-cell transmission of HIV-1 mediated by the major target cells, including dendritic cells, CD4+ T cells, and macrophages. Increasing evidence indicated the significance of host factors and immune responses in HIV-1 mucosal infection and transmission. Summary Recent progress in HIV-1 mucosal infection and transmission enriches our knowledge of virus-host interactions and viral pathogenesis. Functional studies of HIV-1 interactions with host cells can provide new insights into the design of more effective approaches to combat HIV-1 infection and AIDS. PMID:18802490
Michcik, Adam; Michajłowski, Igor; Starzyńska, Anna
Introduction Direct oral microscopy constitutes a novel, non-invasive diagnostic technique, which aids clinical examination of the oral cavity. The oral mucosa is examined at multiple magnifications and features such as sub-epithelial mucosal vessels, surface patterns, colour tone, transparency and the exact demarcation of mucosal lesions are estimated. The incidence of oral squamous cell carcinoma (OSCC) oscillates between 1.9% and 3.5%, which makes it the eighth most common carcinoma occurring around the world and in Poland. The 5-year survival rates oscillate between 20% and 30%. Aim The aim of the study was to evaluate clinically unchanged mucosal margins around OSCC by direct oral microscopy. The authors approached the question whether the borders of mucosal margins around OSCC established via direct oral microscopy differ from those established based on clinical examination. Material and methods Fifteen patients diagnosed with OSCC were enrolled. Patients were first clinically examined to evaluate the extent of the tumour and to plan resection margins. Eventually, direct oral microscopy was performed to establish the width of the subclinically unchanged mucosal margins based on a standard picture of healthy oral mucosae, followed by comparison with those established by clinical evaluation. Results Histopathologic results of biopsies from areas indicated by direct oral microscopy revealed dysplasia in 86.7% of patients, whereas biopsies from areas indicated by clinical examination revealed dysplasia only in 40% of individuals, resulting in the need for widening of mucosal margins. Conclusions Direct oral microscopy enables detection of dysplasia within clinically unaltered mucosal margins around OSCC, which results in more precise establishing of resection boundaries, contributing to improvement of resection totality. PMID:26759543
Fathy, Eman Mahmoud; Shafiek, Hanaa; Morsi, Tamer S; El Sabaa, Bassma; Elnekidy, Abdelaziz; Elhoffy, Mohamed; Atta, Mohamed Samy
Background Bronchial vascular remodeling is an underresearched component of airway remodeling in COPD. Image-enhanced bronchoscopy may offer a less invasive method for studying bronchial microvasculature in COPD. Objectives To evaluate endobronchial mucosal vasculature and changes in COPD by image-enhanced i-scan3 bronchoscopy and correlate them pathologically by analyzing bronchial mucosal biopsies. Methods This case–control study analyzed 29 COPD patients (41.4% Global initiative for chronic Obstructive Lung Disease B [GOLD B] and 58.6% GOLD D) and ten healthy controls admitted at Alexandria Main University Hospital, Egypt. Combined high-definition white light bronchoscopy (HD WLB) with i-scan3 was used to evaluate endobronchial mucosal microvasculature. The vascularity was graded according to the level of mucosal red discoloration (ie, endobronchial erythema) from decreased discoloration to normal, mild, moderate, and severe increased red discoloration (G−1, G0, G+1, G+2, and G+3, respectively) and scored by three bronchoscopists independently. Bronchial mucosal biopsies were taken for microvascular density counting using anti-CD34 antibody as angiogenesis marker. Results Different grades of endobronchial erythema were observed across/within COPD patients using combined HD WLB + i-scan3, with significant agreement among scorers (P=0.031; median score of G+1 [G−1–G+2]) being higher in GOLD D (P=0.001). Endobronchial erythema significantly correlated with COPD duration, exacerbation frequency, and body mass index (P<0.05). Angiogenesis was significantly decreased among COPD patients versus controls (10.6 [8–13.3] vs 14 [11–17.1]; P=0.02). Mucosal surface changes (including edema, atrophy, and nodules) were better visualized by the combined HD WLB + i-scan3 rather than HD WLB alone. Conclusion Combined HD WLB + i-scan3 seems to be valuable in evaluating mucosal microvasculature and surface changes in COPD, which may represent vasodilatation rather than
The ability to obtain information about the structure of tissue without taking a sample for pathology has opened the way for new diagnostic techniques. The present paper reviews all currently available techniques capable of producing an optical biopsy, with or without morphological images. Most of these techniques are carried out by physicians who are not specialized in pathology and therefore not trained to interpret the results as a pathologist would. In these cases, the use of telepathology or distant consultation techniques is essential. PMID:20339507
Kaplan, Ilana; Gal, Gavriel; Chaushu, Gavriel; Allon, Dror M.
Objectives: To investigate the clinical characteristics and pre-biopsy provisional diagnoses of benign oral mucosal tumors. Material and Methods: A 10- year retrospective analysis of all benign tumors of the oral mucosa, from a university- affiliated oral and maxillofacial surgery department. Results: 146 benign tumors were included. The mean age was 49.6 years, with an approximately equal gender distribution. The most prevalent tumor types were lipomatous tumors (27.4%), vascular (23.3%), and salivary gland tumors (16.5%). Tongue, labial and buccal mucosa were the most frequently involved sites. The vast majority (98.6%) presented as non-ulcerated masses. Only 2 (1.4%) presented as ulcerated masses. The clinical provisional diagnosis correctly classified lesions as non-malignant in 93.3%. In only 9 (6.7%) suspicion of malignancy was included in the provisional diagnosis. However, benign neoplasia was unsuspected in 42.1% of tumors. These cases were clinically classified as reactive. Conclusions: Benign tumors were most likely to be clinically correctly classified as non-malignant, but even in the setting of experienced oral surgeons, neoplasia was unsuspected in more than 40% of cases. This data strongly supports the need to biopsy every oral mucosal mass, since inaccurate clinical evaluation of the lesion’s biological nature was a frequent event. Key words:Malignant, benign, reactive, ulcerated mass, non-ulcerated mass, clinical diagnosis. PMID:24316705
Sechi, Leonardo A.; Manuela, Mura; Francesco, Tanda; Amelia, Lissia; Antonello, Solinas; Giovanni, Fadda; Stefania, Zanetti
Crohn's disease is a chronic inflammatory disease of the gastrointestinal tract of unknown etiology. We report on the presence of cell wall-deficient Mycobacterium avium subsp. paratuberculosis in 35 of 48 paraffin-embedded tissue specimens from 33 patients with Crohn's disease by in situ hybridization with IS900 as a probe. PMID:11724871
Mihajlovic, Marija; Vlajkovic, Slobodan; Jovanovic, Predrag; Stefanovic, Vladisav
Primary mucosal melanomas arise from melanocytes located in mucosal membranes lining respiratory, gastrointestinal and urogenital tract. Although a majority of mucosal melanomas originate from the mucosa of the nasal cavity and accessory sinuses, oral cavity, anorectum, vulva and vagina, they can arise in almost any part of mucosal membranes. Most of mucosal melanomas occur in occult sites, which together with the lack of early and specific signs contribute to late diagnosis, and poor prognosis. Because of their rareness the knowledge about their pathogenesis and risk factors is insufficient, and also there are not well established protocols for staging and treatment of mucosal melanomas. Surgery is the mainstay of treatment, with trends toward more conservative treatment since radical surgery did not show an advantage for survival. Radiotherapy can provide better local control in some locations, but did not show improvement in survival. There is no effective systemic therapy for these aggressive tumors. Compared with cutaneous and ocular melanoma, mucosal melanomas have lowest percent of five-year survival. Recently revealed molecular changes underlying mucosal melanomas offer new hope for development of more effective systemic therapy for mucosal melanomas. Herein we presented a comprehensive review of various locations of primary melanoma along mucosal membranes, their epidemiological and clinical features, and treatment options. We also gave a short comparison of some characteristics of cutaneous and mucosal melanomas. PMID:23071856
Teleost fish possess an adaptive immune system associated with each of their mucosal body surfaces. Evidence obtained from mucosal vaccination and mucosal infection studies reveal that adaptive immune responses take place at the different mucosal surfaces of teleost. The main mucosa-associated lymphoid tissues (MALT) of teleosts are the gut-associated lymphoid tissue (GALT), skin-associated lymphoid tissue (SALT), the gill-associated lymphoid tissue (GIALT) and the recently discovered nasopharynx-associated lymphoid tissue (NALT). Teleost MALT includes diffuse B cells and T cells with specific phenotypes different from their systemic counterparts that have co-evolved to defend the microbe-rich mucosal environment. Both B and T cells respond to mucosal infection or vaccination. Specific antibody responses can be measured in the gills, gut and skin mucosal secretions of teleost fish following mucosal infection or vaccination. Rainbow trout studies have shown that IgT antibodies and IgT+ B cells are the predominant B cell subset in all MALT and respond in a compartmentalized manner to mucosal infection. Our current knowledge on adaptive immunity in teleosts is limited compared to the mammalian literature. New research tools and in vivo models are currently being developed in order to help reveal the great intricacy of teleost mucosal adaptive immunity and help improve mucosal vaccination protocols for use in aquaculture. PMID:26274978
Lee, D S; Ahn, Y C; Eom, D W; Lee, S J
Non-Hodgkin lymphoma involving the esophagus is very rare. Only a few cases have been reported in the English literature to date, and it accounts for less than 1% of all cases of gastrointestinal lymphoma. As this malignancy manifests as a submucosal tumor, pathological diagnosis by using a simple endoscopic biopsy alone is difficult. Therefore, surgical biopsy, endoscopic mucosal resection, and endoscopic ultrasound-guided fine-needle aspiration have been used in most cases. Herein, we report a case of esophageal mucosa-associated lymphoid tissue lymphoma in a 49-year-old man, which involved the use of a stacked forceps biopsy to obtain adequate samples for pathological analysis; the use of the stacked forceps biopsy method is unlike those used in previous cases. The patient received cyclophosphamide, vincristine, and prednisolone chemotherapy; he achieved a complete response. In addition, we review the literature relevant to this case.
Sitt, Jacqueline C M; Wong, Priscilla
Ultrasound-guided needle biopsy of synovium is an increasingly performed procedure with a high diagnostic yield. In this review, we discuss the normal synovium, as well as the indications, technique, tissue handling and clinical applications of ultrasound-guided synovial biopsy. PMID:26581578
Mota-Ramírez, Amparo; Silvestre, Francisco Javier; Simó, Juan Manuel
The conclusions drawn from the study of an oral biopsy are considered essential for the definitive diagnosis of diseases of the oral mucosa, and for the subsequent planning of appropriate treatment. Although the obtainment of biopsies is widely used in all medical fields, the practice is not so widespread in dental practice--fundamentally because of a lack of awareness of the procedure among dental professionals. In this context, it must be taken into account that the early diagnosis of invasive oral malignancy may be critical for improving the patient prognosis. However, in some cases the results are adversely affected by incorrect manipulation of the biopsy material. The present study provides an update on the different biopsy sampling techniques and their application. Such familiarization in turn will contribute to knowledge of the material and instruments required for correct biopsy performance in dentistry, as well as of the material required for correct sample storage and transport.
Lendvai, Gabor; Jármay, Katalin; Karácsony, Gizella; Halász, Tünde; Kovalszky, Ilona; Baghy, Kornélia; Wittmann, Tibor; Schaff, Zsuzsa; Kiss, András
AIM: To assess the expression of selected microRNAs (miRNA) in hepatitis C, steatotic hepatitis C, noninfected steatotic and normal liver tissues. METHODS: The relative expression levels of miR-21, miR-33a, miR-96, miR-122, miR-125b, miR-221 and miR-224 were determined in 76 RNA samples isolated from 18 non-steatotic and 28 steatotic chronic hepatitis C (CHC and CHC-Steatosis, respectively) cases, 18 non-infected, steatotic liver biopsies of metabolic origin (Steatosis) and 12 normal formalin-fixed paraffin-embedded liver tissues using TaqMan MicroRNA Assays. All CHC biopsy samples were obtained prior to initiating therapy. Patients’ serum biochemical values, which included glucose, triglyceride, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl-transferase (GGT), alkaline phosphatase (AP), were obtained and correlated with relative miRNA expression. RESULTS: When compared with control non-infected liver samples, miR-122 and miR-221 levels were reduced in CHC-Steatosis (P < 0.03) and in CHC, CHC-Steatosis and Steatosis (P < 0.01). Alternatively, the expression of miR-33a and miR-224 were elevated in CHC-Steatosis and Steatosis in comparison to control tissue (P < 0.01). The levels of miR-33a and miR-224 in CHC-Steatosis (P < 0.02) and miR-224 in Steatosis (P < 0.001) were increased in comparison to CHC samples. By contrast, the expression of miR-21 did not differ statistically between diseased and normal liver samples. Levels of miR-33a correlated negatively with serum AST and AP levels in Steatosis as well as with necroinflammatory grade in CHC, whereas miR-21 correlated positively with AST in Steatosis and displayed negative correlation with triglyceride level in CHC-Steatosis. In contrast, miRNA levels were not correlated with ALT, GGT, cholesterol levels or fibrosis stage. CONCLUSION: Differences in miRNA expression were observed between CHC and steatotic CHC, CHC and steatotic liver, but not between steatotic CHC
Berin, M Cecilia; Sampson, Hugh A
Food allergies are increasing in prevalence at a higher rate than can be explained by genetic factors, suggesting a role for as yet unidentified environmental factors. In this review, we summarize the state of knowledge about the healthy immune response to antigens in the diet and the basis of immune deviation that results in immunoglobulin E (IgE) sensitization and allergic reactivity to foods. The intestinal epithelium forms the interface between the external environment and the mucosal immune system, and emerging data suggest that the interaction between intestinal epithelial cells and mucosal dendritic cells is of particular importance in determining the outcome of immune responses to dietary antigens. Exposure to food allergens through non-oral routes, in particular through the skin, is increasingly recognized as a potentially important factor in the increasing rate of food allergy. There are many open questions on the role of environmental factors, such as dietary factors and microbiota, in the development of food allergy, but data suggest that both have an important modulatory effect on the mucosal immune system. Finally, we discuss recent developments in our understanding of immune mechanisms of clinical manifestations of food allergy. New experimental tools, particularly in the field of genomics and the microbiome, are likely to shed light on factors responsible for the growing clinical problem of food allergy.
Shu, Zhiquan; Levy, Claire N.; Ferre, April L.; Hartig, Heather; Fang, Cifeng; Lentz, Gretchen; Fialkow, Michael; Kirby, Anna C.; Adams Waldorf, Kristina M.; Veazey, Ronald S.; Germann, Anja; von Briesen, Hagen; McElrath, M. Juliana; Dezzutti, Charlene S.; Sinclair, Elizabeth; Baker, Chris A. R.; Shacklett, Barbara L.; Gao, Dayong; Hladik, Florian
Background Understanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible. Methods and Findings To find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10–15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension. Conclusions Leukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes. PMID:27232996
Jalouli, Jamshid; Ibrahim, Salah O; Sapkota, Dipak; Jalouli, Miranda M; Vasstrand, Endre N; Hirsch, Jan M; Larsson, Per-Anders
Using PCR/DNA sequencing, we investigated the prevalence of human papillomavirus (HPV), herpes simplex virus (HSV) and Epstein-Barr virus (EBV) DNA in brush biopsies obtained from 150 users of Sudanese snuff (toombak) and 25 non-users of toombak in formalin-fixed paraffin-embedded tissue samples obtained from 31 patients with oral dysplasias (25 toombak users and 6 non-users), and from 217 patients with oral cancers (145 toombak users and 72 non-users). In the brush tissue samples from toombak users, HPV was detected in 60 (40%), HSV in 44 (29%) and EBV in 97 (65%) of the samples. The corresponding figures for the 25 samples from non-users were 17 (68%) positive for HPV, 6 (24%) positive for HSV and 21 (84%) for EBV. The formalin-fixed samples with oral dysplasias were all negative for HPV. In the 145 oral cancer samples from toombak users, HPV was detected in 39 (27%), HSV in 15 (10%) and EBV in 53 (37%) of the samples. The corresponding figures for the samples from non-users were 15 (21%) positive for HPV, 5 (7%) for HSV and 16 (22%) for EBV. These findings illustrate that prevalence of HSV, HPV and EBV infections are common and may influence oral health and cancer development. It is not obvious that cancer risk is increased in infected toombak users. These observations warrant further studies involving toombak-associated oral lesions, to uncover the possible mechanisms of these viral infections in the development of oral cancer, and the influence of toombak on these viruses.
... A biopsy also helps your doctor determine how aggressive your cancer is — the cancer's grade. The grade ... grade (grade 1) cancers are generally the least aggressive and high-grade (grade 4) cancers are generally ...
Jaszewski, R; Tolia, V; Ehrinpreis, M N; Bodzin, J H; Peleman, R R; Korlipara, R; Weinstock, J V
To define a potential role for the angiotensin system in Crohn's colitis, the colonic mucosal levels of angiotensin I and II were measured in endoscopic biopsy samples from patients with active Crohn's colitis (n = 20), ulcerative colitis (n = 13), other forms of colitis (n = 3), and normal controls (n = 17). Colonic mucosal levels of angiotensin I and II were greater in patients with Crohn's colitis than in normal subjects (p less than 0.001 and p less than 0.001, respectively). Mucosal levels of angiotensin I and II were also higher in Crohn's colitis than in ulcerative colitis (p less than 0.001 and p less than 0.001, respectively), and levels of angiotensin II were higher in Crohn's than in other forms of colitis (p = 0.014). Mucosal levels of angiotensin I and II correlated well with the degree of macroscopic inflammation in Crohn's colitis (r = 0.86, p less than 0.001 and r = 0.68, p less than 0.001, respectively). Mucosal levels of angiotensin I correlated fairly well with the Crohn's Disease Activity Index (r = 0.46, p less than 0.05) while angiotensin II levels correlated poorly. These studies suggest that angiotensin I and II may have a role in the inflammation associated with Crohn's colitis.
Pulmonary biopsy done with a needle of circular bore, can be performed on very young children. A sample of tissue, big enough to establish a precise diagnosis in 2/3 of cases, can be obtained. The main complication is pneumothorax occuring about once in five. Hemoptysia or hemorrhage has never been observed. A proposed indication of premortem biopsy accelerated the death of a child with congenital pulmonary fibrosis. The search of a diagnosis in diffuse pulmonary diseases is the major indication for pulmonary biopsy in the child.
... thinner than the one used for blood tests. Core needle biopsy A core biopsy uses a larger needle to sample breast ... or CT scan. Another way to do a core biopsy is known as vacuum-assisted. Surgical (open) ...
... an excellent technique for making a general, basic identification of the type of bacteria in the sample. ... A.M. Editorial team. Related MedlinePlus Health Topics Bacterial Infections Biopsy Browse the Encyclopedia A.D.A. ...
... Mean Abnormal results may be due to: Stomach (gastric) cancer Gastritis , when the lining of the stomach becomes ... team. Related MedlinePlus Health Topics Biopsy Peptic Ulcer Stomach Cancer Stomach Disorders Browse the Encyclopedia A.D.A. ...
Wullich, B; Füssel, S; Grobholz, R
As individual risk assessment mainly depends on the correct prediction of the tumor's biological behavior, primary diagnosis plays a key role in the clinical management of prostate cancer patients. Prostate core needle biopsy, as a primary diagnostic tool, should not only confirm clinical suspicion but also supply the urologist with information which is necessary for risk-adapted therapy. The experience and competence of both the urologist and the pathologist are crucial for the quality of prostate core needle biopsy diagnosis. Optimized handling and submission of prostate core needle biopsy specimens by the urologist to the pathologist are of outstanding importance for improving the number of cancer cases detected. Increasing availability of molecular markers leads to the necessity of developing new tissue sampling procedures which allow prostate core needle biopsy specimens to be simultaneously studied histologically and by molecular approaches.
Tang, Xian; Chen, Zhiwei
It is well known that mucosal tissues contain the largest surface area of the human body and are the front line of natural host defense against various pathogens. In fact, more than 80% of infectious disease pathogens probably gain entry into the susceptible human hosts through open mucosal surfaces. Human immunodeficiency virus type one (HIV-1), a mainly sexually transmitted virus, also primarily targets the vaginal and gastrointestinal mucosa as entry sites for viral transmission, seeding, replication and amplification. Since HIV-1 establishes its early replication in vaginal or rectal mucosal tissues, the induction of sufficient mucosal immunity at the initial site of HIV-1 transmission becomes essential for a protective vaccine. However, despite the fact that current conventional vaccine strategies have remained unsuccessful in preventing HIV-1 infection, sufficient financial support and resources have yet to be given to develop a vaccine able to elicit protective mucosal immunity against sexual transmissions. Interestingly, Chinese ancestors invented variolation through intranasal administration about one thousand years ago, which led to the discovery of a successful smallpox vaccine and the final eradication of the disease. It is the hope for all mankind that the development of a mucosal AIDS vaccine will ultimately help control the AIDS pandemic. In order to discover an effective mucosal AIDS vaccine, it is necessary to have a deep understanding of mucosal immunology and to test various mucosal vaccination strategies. PMID:21994611
Summers, Paul R
Mucosal yeast infection is best understood as a consequence of compromised mucosal cell-mediated and innate immunity. Defense against oral candidiasis is dominantly cell mediated. The innate immune system may play the main role in regulating vulvovaginal yeast infection. Conditions that compromise cell-mediated immunity such as leukemia, severe illness and HIV infection must be considered as predisposing factors for recurrent oral candidiasis. Compromise of vaginal innate immunity due to mucosal allergy or due to a genetic defect such as mannose-binding lectin deficiency contributes to chronic vulvovaginal yeast infection. Treatment of cofactors must be considered in order to achieve control in recurrent mucosal yeast infection.
Ruas, Ana Cristina Nunes; Lucena, Márcia Mendonça; da Costa, Ananda Dutra; Vieira, Jéssica Rafael; de Araújo-Melo, Maria Helena; Terceiro, Benivaldo Ramos Ferreira; de Sousa Torraca, Tania Salgado; de Oliveira Schubach, Armando; Valete-Rosalino, Claudia Maria
Introduction Leishmaniasis is considered as one of the six most important infectious diseases because of its high detection coefficient and ability to produce deformities. In most cases, mucosal leishmaniasis (ML) occurs as a consequence of cutaneous leishmaniasis. If left untreated, mucosal lesions can leave sequelae, interfering in the swallowing, breathing, voice and speech processes and requiring rehabilitation. Objective To describe the anatomical characteristics and voice quality of ML patients. Materials and Methods A descriptive transversal study was conducted in a cohort of ML patients treated at the Laboratory for Leishmaniasis Surveillance of the Evandro Chagas National Institute of Infectious Diseases - Fiocruz, between 2010 and 2013. The patients were submitted to otorhinolaryngologic clinical examination by endoscopy of the upper airways and digestive tract and to speech-language assessment through directed anamnesis, auditory perception, phonation times and vocal acoustic analysis. The variables of interest were epidemiologic (sex and age) and clinic (lesion location, associated symptoms and voice quality. Results 26 patients under ML treatment and monitored by speech therapists were studied. 21 (81%) were male and five (19%) female, with ages ranging from 15 to 78 years (54.5+15.0 years). The lesions were distributed in the following structures 88.5% nasal, 38.5% oral, 34.6% pharyngeal and 19.2% laryngeal, with some patients presenting lesions in more than one anatomic site. The main complaint was nasal obstruction (73.1%), followed by dysphonia (38.5%), odynophagia (30.8%) and dysphagia (26.9%). 23 patients (84.6%) presented voice quality perturbations. Dysphonia was significantly associated to lesions in the larynx, pharynx and oral cavity. Conclusion We observed that vocal quality perturbations are frequent in patients with mucosal leishmaniasis, even without laryngeal lesions; they are probably associated to disorders of some resonance
Suter, Melissa; Reinhardt, Joseph M.; Riker, David; Ferguson, John Scott; McLennan, Geoffrey
Airway mucosal color changes occur in response to the development of bronchial diseases including lung cancer, cystic fibrosis, chronic bronchitis, emphysema and asthma. These associated changes are often visualized using standard macro-optical bronchoscopy techniques. A limitation to this form of assessment is that the subtle changes that indicate early stages in disease development may often be missed as a result of this highly subjective assessment, especially in inexperienced bronchoscopists. Tri-chromatic CCD chip bronchoscopes allow for digital color analysis of the pulmonary airway mucosa. This form of analysis may facilitate a greater understanding of airway disease response. A 2-step image classification approach is employed: the first step is to distinguish between healthy and diseased bronchoscope images and the second is to classify the detected abnormal images into 1 of 4 possible disease categories. A database of airway mucosal color constructed from healthy human volunteers is used as a standard against which statistical comparisons are made from mucosa with known apparent airway abnormalities. This approach demonstrates great promise as an effective detection and diagnosis tool to highlight potentially abnormal airway mucosa identifying a region possibly suited to further analysis via airway forceps biopsy, or newly developed micro-optical biopsy strategies. Following the identification of abnormal airway images a neural network is used to distinguish between the different disease classes. We have shown that classification of potentially diseased airway mucosa is possible through comparative color analysis of digital bronchoscope images. The combination of the two strategies appears to increase the classification accuracy in addition to greatly decreasing the computational time.
Cassmann, Eric; White, Robin; Atherly, Todd; Wang, Chong; Sun, Yaxuan; Khoda, Samir; Mosher, Curtis; Ackermann, Mark; Jergens, Albert
Background The intestinal microbiota is increasingly linked to the pathogenesis of chronic enteropathies (CE) in dogs. While imbalances in duodenal and fecal microbial communities have been associated with mucosal inflammation, relatively little is known about alterations in mucosal bacteria seen with CE involving the ileum and colon. Aim To investigate the composition and spatial organization of mucosal microbiota in dogs with CE and controls. Methods Tissue sections from endoscopic biopsies of the ileum and colon from 19 dogs with inflammatory bowel disease (IBD), 6 dogs with granulomatous colitis (GC), 12 dogs with intestinal neoplasia, and 15 controls were studied by fluorescence in situ hybridization (FISH) on a quantifiable basis. Results The ileal and colonic mucosa of healthy dogs and dogs with CE is predominantly colonized by bacteria localized to free and adherent mucus compartments. CE dogs harbored more (P < 0.05) mucosal bacteria belonging to the Clostridium-coccoides/Eubacterium rectale group, Bacteroides, Enterobacteriaceae, and Escherichia coli versus controls. Within the CE group, IBD dogs had increased (P < 0.05) Enterobacteriaceae and E. coli bacteria attached onto surface epithelia or invading within the intestinal mucosa. Bacterial invasion with E. coli was observed in the ileal and colonic mucosa of dogs with GC (P < 0.05). Dogs with intestinal neoplasia had increased (P < 0.05) adherent (total bacteria, Enterobacteriaceae, E. coli) and invasive (Enterobacteriaceae, E. coli, and Bacteroides) bacteria in biopsy specimens. Increased numbers of total bacteria adherent to the colonic mucosa were associated with clinical disease severity in IBD dogs (P < 0.05). Conclusion Pathogenic events in canine CE are associated with different populations of the ileal and colonic mucosal microbiota. PMID:26840462
Pinto-Sánchez, María Inés; Nachman, Fabio D.; Fuxman, Claudia; Iantorno, Guido; Hwang, Hui Jer; Ditaranto, Andrés; Costa, Florencia; Longarini, Gabriela; Wang, Xuan Yu; Huang, Xianxi; Vázquez, Horacio; Moreno, María L.; Niveloni, Sonia; Bercik, Premysl; Smecuol, Edgardo; Mazure, Roberto; Bilder, Claudio; Mauriño, Eduardo C.; Verdu, Elena F.; Bai, Julio C.
Background/Aim. Reflux symptoms (RS) are common in patients with celiac disease (CD), a chronic enteropathy that affects primarily the small intestine. We evaluated mucosal integrity and motility of the lower esophagus as mechanisms contributing to RS generation in patients with CD. Methods. We enrolled newly diagnosed CD patients with and without RS, nonceliac patients with classical reflux disease (GERD), and controls (without RS). Endoscopic biopsies from the distal esophagus were assessed for dilated intercellular space (DIS) by light microscopy and electron microscopy. Tight junction (TJ) mRNA proteins expression for zonula occludens-1 (ZO-1) and claudin-2 and claudin-3 (CLDN-2; CLDN-3) was determined using qRT-PCR. Results. DIS scores were higher in patients with active CD than in controls, but similar to GERD patients. The altered DIS was found even in CD patients without RS and normalized after one year of a gluten-free diet. CD patients with and without RS had lower expression of ZO-1 than controls. The expression of CLDN-2 and CLDN-3 was similar in CD and GERD patients. Conclusions. Our study shows that patients with active CD have altered esophageal mucosal integrity, independently of the presence of RS. The altered expression of ZO-1 may underlie loss of TJ integrity in the esophageal mucosa and may contribute to RS generation. PMID:27446827
Lazado, Carlo C; Caipang, Christopher Marlowe A
Teleost mucosal immunity has become the subject of unprecedented research studies in recent years because of its diversity and defining characteristics. Its immune repertoire is governed by the mucosa-associated lymphoid tissues (MALT) which are divided into gut-associated lymphoid tissues (GALT), skin-associated lymphoid tissues (SALT), and gill-associated lymphoid tissues (GIALT). The direct contact with its immediate environment makes the mucosal surfaces of fish susceptible to a wide variety of pathogens. The inherent immunocompetent cells and factors in the mucosal surfaces together with the commensal microbiota have pivotal role against pathogens. Immunomodulation is a popular prophylactic strategy in teleost and probiotics possess this beneficial feature. Most of the studies on the immunomodulatory properties of probiotics in fish mainly discussed their impacts on systemic immunity. In contrast, few of these studies discussed the immunomodulatory features of probiotics in mucosal surfaces and are concentrated on the influences in the gut. Significant attention should be devoted in understanding the relationship of mucosal immunity and probiotics as the present knowledge is limited and are mostly based on extrapolations of studies in humans and terrestrial vertebrates. In the course of the advancement of mucosal immunity and probiotics, new perspectives in probiotics research, e.g., probiogenomics have emerged. This review affirms the relevance of probiotics in the mucosal immunity of fish by revisiting and bridging the current knowledge on teleost mucosal immunity, mucosal microbiota and immunomodulation of mucosal surfaces by probiotics. Expanding the knowledge of immunomodulatory properties of probiotics especially on mucosal immunity is essential in advancing the use of probiotics as a sustainable and viable strategy for successful fish husbandry.
Recent progress in cancer biology has revealed the fact that molecular profiles of primary and metastatic cancer are not necessarily the same. Furthermore, evidence of intra-tumor heterogeneity has been disclosed repeatedly. In addition to these, acquiring resistances to chemoradiation therapy is far more rapid than typical predictions. Under these circumstances, physicians are realizing that one biopsy is not enough to predict the direction of cancer progression or extension. Repeated biopsy was proposed in this context. For "re-biopsy", acquiring blood is much easier compared to regular biopsies of acquiring body tissues. Therefore, CTC or Cell-free DNA is one of the hot topics in clinical and molecular diagnostic fields. The term "liquid biopsy" is used to include these two materials. We utilized a CTC isolation device based on microfluidic principles. Procedures for the extraction of DNA from plasma (Cell-free DNA) is also available. Based on this background, we performed a feasibility study of NGS (Next Generation Sequencing) by analyzing materials from advanced gastrointestinal cancer patients. We have successfully acquired NGS results using these liquid biopsies. We have also investigated the possibility of storing CTCs by evaluating procedures after cytospin using H1975 cells with various fixation conditions under a DIC microscope examination. Because of the paucity of the number of isolated CTCs, H1975 cells were used for this purpose. After cytospin, 95% ETOH and then -80 degrees C storage provided the best results. Attempts at not only NGS but also storage in this sequence of studies have opened new fields of liquid biopsy in clinical laboratories.
Kleessen, Brigitta; Blaut, Michael
Non-digestible inulin-type fructans, such as oligofructose and high-molecular-weight inulin, have been shown to have the ability to alter the intestinal microbiota composition in such a way that members of the microbial community, generally considered as health-promoting, are stimulated. Bifidobacteria and lactobacilli are the most frequently targeted organisms. Less information exists on effects of inulin-type fructans on the composition, metabolism and health-related significance of bacteria at or near the mucosa surface or in the mucus layer forming mucosa-associated biofilms. Using rats inoculated with a human faecal flora as an experimental model we have found that inulin-type fructans in the diet modulated the gut microbiota by stimulation of mucosa-associated bifidobacteria as well as by partial reduction of pathogenic Salmonella enterica subsp. enterica serovar Typhimurium and thereby benefit health. In addition to changes in mucosal biofilms, inulin-type fructans also induced changes in the colonic mucosa stimulating proliferation in the crypts, increasing the release of mucins, and altering the profile of mucin components in the goblet cells and epithelial mucus layer. These results indicate that inulin-type fructans may stabilise the gut mucosal barrier. Dietary supplementation with these prebiotics could offer a new approach to supporting the barrier function of the mucosa.
Barasch, Andrei; Elad, Sharon; Altman, Arnold; Damato, Kathryn; Epstein, Joel
This review focuses on the value of several groups of agents for the prevention and treatment of mucositis. The review refers to alimentary mucositis as a generalized term that includes oral mucositis and gastrointestinal mucositis. This paper is part of the systematic review made by the mucositis study group which operates in the Multinational Association of Supportive Care in Cancer (MASCC)/International Society of Oral Oncology (ISOO). Several new guidelines are suggested in this review as an update to the primary systematic review that was published by the same group in 2004.
Jain, Vk; Jindal, N; Imchen, S
Pemphigus vegetans is a rare variant of pemphigus vulgaris. A 62-year-old woman presented with erythematous moist vegetative plaque on the left breast and left groin. There was no mucosal involvement. Histopathological and direct immunofluorescence findings were suggestive of pemphigus vegetans. She showed excellent response to oral steroids. Literature is scarcely available on the limited involvement with pemphigus vegetans without mucosal involvement.
Gómez, Daniela; Bartholomew, Jerri; Sunyer, J. Oriol
Myxozoans are among the most abundant parasites in nature. Their life cycles involve two hosts: an invertebrate, usually an annelid, and a vertebrate, usually a fish. They affect fish species in their natural habitats but also constitute a menace for fish aquaculture. Using different strategies they are able to parasitize and cause damage in multiple organs, including mucosal tissues, which they use also as portals of entry. In fish, the main mucosal sites include the intestine, skin and gills. Recently the finding of a specific mucosal immunoglobulin in teleost (IgT), analogous to mammalian IgA, and the capacity of fish to develop a specific mucosal immune response against different pathogens, has highlighted the importance of studying immune responses at mucosal sites. In this review, we describe the major biological characteristics of myxozoan parasites and present the data available regarding immune responses for species that infect mucosal sites. As models for mucosal immunity we review the responses to Enteromyxum spp. and Ceratomyxa shasta, both of which parasitize the intestine. The immune response at the skin and gills is also described, as these mucosal tissues are used by myxozoans as attaching surfaces and portal of entry, and some species also parasitize these sites. Finally, the development of immunoprophylactic strategies is discussed. PMID:23994774
Krausert, Christopher R.; Olszewski, Aleksandra E.; Taylor, Lindsay N.; McMurray, James S.; Dailey, Seth H.; Jiang, Jack J.
Organized vibration of the vocal folds is critical to high quality voice production. When the vocal folds oscillate, the superficial tissue of the vocal fold is displaced in a wave-like fashion, creating the so called “mucosal wave”. Because the mucosal wave is dependent on vocal fold structure, physical alterations of that structure cause mucosal wave abnormalities. Visualization and quantification of mucosal wave properties have become useful parameters in diagnosing and managing vocal fold pathology. Mucosal wave measurement provides information about vocal fold characteristics that cannot be determined with other assessment techniques. Here, we discuss the benefits, disadvantages, and clinical applicability of the different mucosal wave measurement techniques, such as electroglottography (EGG), photoglottography (PGG), and ultrasound and visualization techniques that include videokymography (VKG), stroboscopy, and high-speed digital imaging (HSDI). The various techniques and their specific uses are reviewed with the intention of helping researchers and clinicians choose a method for a given situation and understand its limitations as well as its potential applications. Recent applications of these techniques for quantitative assessment demonstrate that additional research must be conducted to realize the full potential of these tools. Evaluations of existing research and recommendations for future research are given to promote both the quantitative study of the mucosal wave through accurate and standardized measurement of mucosal wave parameters and the development of reliable methods with which physicians can diagnose vocal disorders. PMID:20471798
Ogra, Pearay L.; Faden, Howard; Welliver, Robert C.
Mucosal administration of vaccines is an important approach to the induction of appropriate immune responses to microbial and other environmental antigens in systemic sites and peripheral blood as well as in most external mucosal surfaces. The development of specific antibody- or T-cell-mediated immunologic responses and the induction of mucosally induced systemic immunologic hyporesponsiveness (oral or mucosal tolerance) depend on complex sets of immunologic events, including the nature of the antigenic stimulation of specialized lymphoid structures in the host, antigen-induced activation of different populations of regulatory T cells (Th1 versus Th2), and the expression of proinflammatory and immunoregulatory cytokines. Availability of mucosal vaccines will provide a painless approach to deliver large numbers of vaccine antigens for human immunization. Currently, an average infant will receive 20 to 25 percutaneous injections for vaccination against different childhood infections by 18 months of age. It should be possible to develop for human use effective, nonliving, recombinant, replicating, transgenic, and microbial vector- or plant-based mucosal vaccines to prevent infections. Based on the experience with many dietary antigens, it is also possible to manipulate the mucosal immune system to induce systemic tolerance against environmental, dietary, and possibly other autoantigens associated with allergic and autoimmune disorders. Mucosal immunity offers new strategies to induce protective immune responses against a variety of infectious agents. Such immunization may also provide new prophylactic or therapeutic avenues in the control of autoimmune diseases in humans. PMID:11292646
Sato, Shintaro; Kiyono, Hiroshi; Fujihashi, Kohtaro
It has been shown that pathogen-specific secretory IgA (SIgA) antibody (Ab) is the major player at mucosal surfaces for host defense. However, alterations in the mucosal immune system occur in advanced aging which results in a failure of induction of SIgA Abs for protection from infectious diseases. Signs of mucosal senescence first appear in the gut immune system. Further, changes in the intestinal microbiota most likely influence mucosal immunity. To overcome the immunological aging decline in mucosal immunity, several adjuvant systems including mucosal dendritic cell (DC) targeting have been shown to be attractive and effective immunological strategies. Similarly, antigen (Ag) uptake-M cells are ideal targets for facilitating Ag-specific mucosal immune responses. However, the numbers of M cells are reduced in aged mice. In this regard, Spi-B, an essential transcription factor for the functional and structural differentiation of M cells could be a potent strategy for the induction of effective mucosal immunity in aging. PMID:25531743
Srivastava, Atul; Gowda, Devegowda Vishakante; Madhunapantula, SubbaRao V; Shinde, Chetan G; Iyer, Meenakshi
Mucosal immune responses are the first-line defensive mechanisms against a variety of infections. Therefore, immunizations of mucosal surfaces from which majority of infectious agents make their entry, helps to protect the body against infections. Hence, vaccinization of mucosal surfaces by using mucosal vaccines provides the basis for generating protective immunity both in the mucosal and systemic immune compartments. Mucosal vaccines offer several advantages over parenteral immunization. For example, (i) ease of administration; (ii) non-invasiveness; (iii) high-patient compliance; and (iv) suitability for mass vaccination. Despite these benefits, to date, only very few mucosal vaccines have been developed using whole microorganisms and approved for use in humans. This is due to various challenges associated with the development of an effective mucosal vaccine that can work against a variety of infections, and various problems concerned with the safe delivery of developed vaccine. For instance, protein antigen alone is not just sufficient enough for the optimal delivery of antigen(s) mucosally. Hence, efforts have been made to develop better prophylactic and therapeutic vaccines for improved mucosal Th1 and Th2 immune responses using an efficient and safe immunostimulatory molecule and novel delivery carriers. Therefore, in this review, we have made an attempt to cover the recent advancements in the development of adjuvants and delivery carriers for safe and effective mucosal vaccine production.
Nizard, Mevyn; Diniz, Mariana O; Roussel, Helene; Tran, Thi; Ferreira, Luis Cs; Badoual, Cecile; Tartour, Eric
The mucosal immune system displays several adaptations reflecting the exposure to the external environment. The efficient induction of mucosal immune responses also requires specific approaches, such as the use of appropriate administration routes and specific adjuvants and/or delivery systems. In contrast to vaccines delivered via parenteral routes, experimental, and clinical evidences demonstrated that mucosal vaccines can efficiently induce local immune responses to pathogens or tumors located at mucosal sites as well as systemic response. At least in part, such features can be explained by the compartmentalization of mucosal B and T cell populations that play important roles in the modulation of local immune responses. In the present review, we discuss molecular and cellular features of the mucosal immune system as well as novel immunization approaches that may lead to the development of innovative and efficient vaccines targeting pathogens and tumors at different mucosal sites.
Yilmaz, Nergiz; Nisbet, Ozlem; Nisbet, Cevat; Ceylan, Gözlem; Hoşgör, Fatih; Dede, O Doğu
The aim of this study was to describe and quantify the therapeutic value of honey in oral mucosal ulcers healing in comparison with Glyceroloxytriester (TGO). We also aimed to biochemically evaluate the healing effects of honey which had been collected from the Blacksea region flora on mucosal ulcers resulting in material loss.Thirty wistar rats (240+/-30 g) were enrolled in this study. Excisional wounds were performed in all rats for animal oral mucosal ulcer model. They were randomly allocated to three groups: group 1 was treated with Apitherapeutic agent or honey (0,1 ml, 2x1), group 2 was treated with TGO (0,1ml, 2x1) locally, Group 3 served as the control group.Following the surgical procedure on day 7, biopsy specimens were taken from right buccal mucosa and on day 14 biopsy specimens were taken from left bucal mucosa in all rats. Afterwards, hydroxy pyroline levels were measured. Data were analyzed statistically.There was no statistically significant difference between Group 1 and 2, and also between Group 2 and 3, but there was statistically significant difference between Group 1 and 3 on day 7. There was no statistically significant difference between Group 1, 2 and 3 on day 14.
Keefe, Dorothy M K
Mucositis is an important toxicity to be aware of in anticancer therapy. It contributes to a reduction in cure rates from cancer. Until recently, it has been poorly understood and therefore has not been well managed. It causes patient distress, delays in treatment administration, and reductions in dose intensity, and it costs the health-care system a large amount of money. Mucositis has traditionally been associated more with hematologic malignancies than with solid tumors, because the incidence of severe mucositis has been much higher with the high-dose chemotherapy regimens used in hematologic malignancies. However, the chemotherapy used in solid tumors also causes mucositis and deserves further study. The separation between oral and gastrointestinal mucositis is potentially false and is being removed, with much research now investigating the entire alimentary canal. There are similarities and differences between radiation therapy- and chemotherapy-induced mucositis, and these have implications for treatment and prevention scheduling and type. Risk prediction is another area that requires more work, but there is real hope that, in the future, we might be able to predict who will suffer from mucositis and in which parts of the alimentary canal, thus enabling us to appropriately target the newer antimucotoxic therapies. The Mucositis Study Goup of the Multinational Association for Supportive Care in Cancer has recently published management guidelines for oral and gastrointestinal mucositis and is in the process of updating them. The guidelines serve as an excellent starting place for future mucositis research because they not only review the available treatments but also discuss mechanisms and epidemiology.
Spraker, Terry R; VerCauteren, Kurt C; Gidlewski, Thomas; Schneider, David A; Munger, Randy; Balachandran, Aru; O'Rourke, Katherine I
Antemortem biopsy of the rectal mucosa was evaluated as a method for the preclinical diagnosis of chronic wasting disease (CWD) in a herd of ranch-raised Rocky Mountain elk (Cervus elaphus nelsoni) quarantined because of exposure to CWD. Biopsy samples were obtained from 41 elk during the winter of 2005-2006 and from 26 elk from that herd still alive and available for testing during the winter of 2006-2007. Samples were examined for PrP(CWD), the protein marker for CWD infection, by immunohistochemistry. PrP(CWD) was detected in follicles of the rectoanal mucosa-associated lymphoid tissue in biopsy samples from 1 elk with clinical signs of chronic wasting disease and 5 clinically normal elk. The diagnosis was confirmed in all 6 animals by postmortem analysis of brain and peripheral lymph nodes. PrP(CWD) was also observed in the submucosal plexus and myenteric plexus of the enteric nervous system, and in close association with nonmyelinated mucosal and submucosal nerve fibers. In antemortem rectal biopsy samples from positive animals, immunostaining was consistently observed in approximately 60% of the mucosa-associated lymphoid tissue follicles if 10 or more total follicles per biopsy were present for evaluation. Most antemortem biopsy samples obtained from elk younger than 6.5 years contained at least 10 follicles per rectal mucosal biopsy. These findings support the analysis of antemortem biopsy of the rectal mucosa samples as part of an integrated strategy to manage chronic wasting disease in Rocky Mountain elk.
Haifler, Miki; Kutikov, Alexander
Renal masses are diagnosed with an increasing frequency. However, a significant proportion of these masses are benign, and the majority of malignant tumors are biologically indolent. Furthermore, renal tumors are often harbored by the elderly and comorbid patients. As such, matching of renal tumor biology to appropriate treatment intensity is an urgent clinical need. Renal mass biopsy is currently a very useful clinical tool that can assist with critical clinical decision-making in patients with renal mass. Yet, renal mass biopsy is associated with limitations and, as such, may not be appropriate for all patients.
Ochiai, T.; Yasutake, W.T.; Gould, R.W.
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.
Tanić, Miljana; Yanowski, Kira; Andrés, Eduardo; Gómez-López, Gonzalo; Socorro, María Rodríguez-Pinilla; Pisano, David G.; Martinez-Delgado, Beatriz; Benítez, Javier
Hereditary breast cancer constitutes only 5–10% of all breast cancer cases and is characterized by strong family history of breast and/or other associated cancer types. Only ~ 25% of hereditary breast cancer cases carry a mutation in BRCA1 or BRCA2 gene, while mutations in other rare high and moderate-risk genes and common low penetrance variants may account for additional 20% of the cases. Thus the majority of cases are still unaccounted for and designated as BRCAX tumors. MicroRNAs are small non-coding RNAs that play important roles as regulators of gene expression and are deregulated in cancer. To characterize hereditary breast tumors based on their miRNA expression profiles we performed global microarray miRNA expression profiling on a retrospective cohort of 80 FFPE breast tissues, including 66 hereditary breast tumors (13 BRCA1, 10 BRCA2 and 43 BRCAX), 10 sporadic breast carcinomas and 4 normal breast tissues, using Exiqon miRCURY LNA™ microRNA Array v.11.0. Here we describe in detail the miRNA microarray expression data and tumor samples used for the study of BRCAX tumor heterogeneity (Tanic et al., 2013) and biomarkers associated with positive BRCA1/2 mutation status (Tanic et al., 2014). Additionally, we provide the R code for data preprocessing and quality control. PMID:26484152
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...
Shaw, Darcy; Gohil, Kartik; Basson, Marc D
Mucosal adaptation is an essential process in gut homeostasis. The intestinal mucosa adapts to a range of pathological conditions including starvation, short-gut syndrome, obesity, and bariatric surgery. Broadly, these adaptive functions can be grouped into proliferation and differentiation. These are influenced by diverse interactions with hormonal, immune, dietary, nervous, and mechanical stimuli. It seems likely that clinical outcomes can be improved by manipulating the physiology of adaptation. This review will summarize current understanding of the basic science surrounding adaptation, delineate the wide range of potential targets for therapeutic intervention, and discuss how these might be incorporated into an overall treatment plan. Deeper insight into the physiologic basis of adaptation will identify further targets for intervention to improve clinical outcomes.
Shaw, Darcy; Gohil, Kartik; Basson, Marc D
Mucosal adaptation is an essential process in gut homeostasis. The intestinal mucosa adapts to a range of pathological conditions including starvation, short-gut syndrome, obesity, and bariatric surgery. Broadly, these adaptive functions can be grouped into proliferation and differentiation. These are influenced by diverse interactions with hormonal, immune, dietary, nervous, and mechanical stimuli. It seems likely that clinical outcomes can be improved by manipulating the physiology of adaptation. This review will summarize current understanding of the basic science surrounding adaptation, delineate the wide range of potential targets for therapeutic intervention, and discuss how these might be incorporated into an overall treatment plan. Deeper insight into the physiologic basis of adaptation will identify further targets for intervention to improve clinical outcomes. PMID:23197881
Gaze, Soraya; McSorley, Henry J; Daveson, James; Jones, Di; Bethony, Jeffrey M; Oliveira, Luciana M; Speare, Richard; McCarthy, James S; Engwerda, Christian R; Croese, John; Loukas, Alex
The mucosal cytokine response of healthy humans to parasitic helminths has never been reported. We investigated the systemic and mucosal cytokine responses to hookworm infection in experimentally infected, previously hookworm naive individuals from non-endemic areas. We collected both peripheral blood and duodenal biopsies to assess the systemic immune response, as well as the response at the site of adult worm establishment. Our results show that experimental hookworm infection leads to a strong systemic and mucosal Th2 (IL-4, IL-5, IL-9 and IL-13) and regulatory (IL-10 and TGF-β) response, with some evidence of a Th1 (IFN-γ and IL-2) response. Despite upregulation after patency of both IL-15 and ALDH1A2, a known Th17-inducing combination in inflammatory diseases, we saw no evidence of a Th17 (IL-17) response. Moreover, we observed strong suppression of mucosal IL-23 and upregulation of IL-22 during established hookworm infection, suggesting a potential mechanism by which Th17 responses are suppressed, and highlighting the potential that hookworms and their secreted proteins offer as therapeutics for human inflammatory diseases.
Cohen, Fernand S; Taslidere, Ezgi; Murthy, Sreekant
In this paper we present a technique to raise a flag on the fly when a transition occurs between different mucosal architectures on or below the surface. The segmentation is based on a novel difference metric for detecting an abrupt change in the parameters extracted from a Stochastic Decomposition Method (SDM) that models the scattered light reflected from the mucosal tissue structure over an area (2-D scan) illuminated by an optical sensor (fiber) emitting light at either one wavelength or with white light. This work has the potential to enhance the endoscopist's ability to locate and identify abnormal mucosal architectures in particular when the disease is developing below the surface and hence becoming hidden during colonoscopy or endoscopic examination. It also has also potential in helping deciding as to when and where to take biopsies; steps that should lead to improvement in the diagnostic yield.
Funatsu, Toshiyuki; Chono, Koji; Hirata, Takuya; Keto, Yoshihiro; Kimoto, Aishi; Sasamata, Masao
The mechanism by which nonsteroidal anti-inflammatory drugs (NSAIDs) suppress gastric mucosal blood flow is not fully understood, although the depletion of mucosal prostaglandin E2 has been proposed as one possible explanation. We investigated the role of gastric acid on gastric mucosal blood flow in NSAID-treated rats. A rat stomach was mounted in an ex vivo chamber, and gastric mucosal blood flow was measured sequentially in a 5-mm2 area of the gastric corpus using a scanning laser Doppler perfusion image system. Results showed that diclofenac (5 mg/kg s.c.) and indomethacin (10 mg/kg s.c.) did not affect gastric mucosal blood flow, although both strongly decreased mucosal prostaglandin E2 when saline was instilled into the gastric chamber. On replacement of the saline in the chamber with 100 mM hydrochloric acid, these drugs caused a decrease in gastric mucosal blood flow levels within 30 min. The specific cyclooxygenase (COX)-2 inhibitors celecoxib (50 mg/kg s.c.) and rofecoxib (25 mg/kg s.c.) did not affect mucosal prostaglandin E2 level, nor did they decrease gastric mucosal blood flow, even when hydrochloric acid was added to the chamber. Furthermore, measurement of vasoconstrictive factors present in the mucosa showed that endothelin-1 levels increased after administration of diclofenac s.c. in the presence of intragastric hydrochloric acid. This indicates that the presence of mucosal hydrochloric acid plays an important role in the NSAID-induced decrease in gastric mucosal blood flow, while the COX-1-derived basal prostaglandin E2, which is unlikely to control gastric mucosal blood flow itself, protects microcirculatory systems from mucosal hydrochloric acid.
A syringe-like disposable cutaneous biopsy instrument equipped with a tubular blade at its lower end, and designed so that a vacuum is created during use, said vacuum serving to retain undeformed a plug of tissue cut from a patient's skin.
Rishi, Pukhraj; Dhami, Abhinav; Biswas, Jyotirmay
Biopsy involves the surgical removal of a tissue specimen for histopathologic evaluation. Most intraocular tumors are reliably diagnosed based on the clinical evaluation or with noninvasive diagnostic techniques. However, accurately diagnosing a small percentage of tumors can be challenging. A tissue biopsy is thus needed to establish a definitive diagnosis and plan the requisite treatment. From fine-needle aspiration biopsy (FNAB) to surgical excision, all tissue collection techniques have been studied in the literature. Each technique has its indications and limitations. FNAB has been reported to provide for 88–95% reliable and safe ophthalmic tumor diagnosis and has gained popularity for prognostic purposes and providing eye conserving treatment surgeries. The technique and instrumentation for biopsy vary depending upon the tissue involved (retina, choroid, subretinal space, vitreous, and aqueous), suspected diagnosis, size, location, associated retinal detachment, and clarity of the media. The cytopathologist confers a very important role in diagnosis and their assistance plays a key role in managing and planning the treatment for malignancies. PMID:27488148
A syringe-like disposable cutaneous biopsy instrument equipped with a tubular blade at its lower end, and designed so that a vacuum is created during use, said vacuum serving to retain undeformed a plug of tissue cut from a patient's skin.
Norling, Karin; Bally, Marta; Höök, Fredrik
Immune protection against infectious diseases is most effective if located at the portal of entry of the pathogen. Hence, there is an increasing demand for vaccine formulations that can induce strong protective immunity following oral, respiratory, or genital tract administration. At present, only few mucosal vaccines are found on the market, but recent technological advancements and a better understanding of the principles that govern priming of mucosal immune responses have contributed to a more optimistic view on the future of mucosal vaccines. Compared to live attenuated vaccines, subcomponent vaccines, most often protein-based, are considered safer, more stable, and less complicated to manufacture, but they require the addition of nontoxic and clinically safe adjuvants to be effective. In addition, another limiting factor is the large antigen dose that usually is required for mucosal vaccines. Therefore, the combination of mucosal adjuvants with the recent progress in nanoparticle technology provides an attractive solution to these problems. In particular, the liposome technology is ideal for combining protein antigen and adjuvant into an effective mucosal vaccine. Here, we describe and discuss recent progress in nanoparticle formulations using various types of liposomes that convey strong promise for the successful development of the next generation of mucosal vaccines. PMID:28127567
Bagán-Sebastián, José V
Introduction The management of oral mucositis is a challenge, due to its complex biological nature. Over the last 10 years, different strategies have been developed for the management of oral mucositis caused by chemotherapy in cancer patients. Material and Methods An exhaustive search was made of the PubMed-Medline, Cochrane Library and Scopus databases, crossing the key words “oral mucositis”, “prevention” and “treatment” with the terms “chemotherapy” and “radiotherapy” by means of the boolean operators “AND” and “NOT”. A total of 268 articles were obtained, of which 96 met the inclusion criteria. Results Several interventions for the prevention of oral mucositis, such as oral hygiene protocols, amifostine, benzidamine, calcium phosphate, cryotherapy and iseganan, among others, were found to yield only limited benefits. Other studies have reported a decrease in the appearance and severity of mucositis with the use of cytoprotectors (sucralfate, oral glutamine, hyaluronic acid), growth factors, topical polyvinylpyrrolidone, and low power laser irradiation. Conclusions Very few interventions of confirmed efficacy are available for the management of oral mucositis due to chemotherapy. However, according to the reviewed literature, the use of palifermin, cryotherapy and low power laser offers benefits, reducing the incidence and severity of oral mucositis – though further studies are needed to confirm the results obtained. Key words:Chemotherapy-Induced Oral Mucositis Treatment. PMID:27034762
Bowen, Joanne M; Gibson, Rachel J; Keefe, Dorothy M K
Alimentary mucositis is a major acute complication in the clinical setting, occurring in a large percentage of patients undergoing cytotoxic therapy. One of the major problems with alimentary mucositis is that the underlying mechanisms behind its development are not entirely understood, which makes it extremely difficult to develop effective interventions. Animal models provide a critical source of knowledge when sampling from patients is unavailable or interventions are yet to be fully tested. This review focuses on the animal models used to increase our understanding of the mechanisms of mucositis and translate new antimucotoxic agents into clinical trials.
Rothwell, B.R.; Spektor, W.S.
Oral mucositis associated with head and neck radiotherapy can substantially hinder completion of cancer therapy. Alleviation of this often severe stomatitis can provide enhanced patient comfort and facilitate appropriate care. A double-blind format was used in a pilot project to measure, against a control rinse, the effectiveness of an oral rinse consisting of hydrocortisone, nystatin, tetracycline, and diphenhydramine in controlling radiation-related mucositis. A combination of clinical evaluation and patient responses to a questionnaire was used to judge the results of the topical medications. Patients using the experimental medication developed less mucositis than did patients in the control group.
Emerick, Kevin S.; Kraft, Stefan; Holbrook, Eric H.
Background: The progression from a benign pigmented lesion on the skin to cutaneous melanoma is better understood, and it could be presumed that a similar progression occurs with mucosal lesions. However, to our knowledge, there has never been documentation of melanosis transforming into melanoma over time. Objective: To describe a transformation of a mucosal melanosis into melanoma. Methods: A 53-year-old man with diffuse melanosis of the nasal cavity underwent surgical resection. Results: Pathology revealed melanocytic hyperplasia without evidence of melanoma. The patient was serially examined, with excisions for new areas of melanosis. The pathology progressed to severely atypical melanocytic proliferation and melanoma in situ over a 4-year period. Conclusion: Nasal melanosis may be a precancerous lesion and may transform into melanoma. All melanosis should be biopsied with close endoscopic observation. Lesions with dysplasia or atypia should be excised due to potential transformation to melanoma. PMID:28107150
Barr, R G; Watkins, J B; Perman, J A
To evaluate the role of the lactose breath hydrogen test for the detection of lactose malabsorption in children with chronic nonspecific abdominal complaints, breath hydrogen excretion was measured in 131 children with recurrent abdominal pain (n = 75) or chronic nonspecific diarrhea (n = 56) following a lactose load (2 gm/kg; maximum 50 gm). The data were compared to those obtained from lactose tolerance tests (n = 113) and symptom response following a lactose load (n = 109) performed simultaneously with the lactose breath hydrogen test, and with results from small bowel biopsies obtained in 31 children to determine dissacharidase activity and mucosal histology. The results indicate that an increase in breath hydrogen of greater than 10 ppm above base line values (delta ppm) by 120 minutes ("early increase" response) completely discriminates between biopsy-proven isolated lactase-insufficient and lactase-sufficient children. A similar increase after 120 minutes ("late increase" response) is consistent both with normal mucosal function and partial lactase insufficiency due to mucosal injury. Breath hydrogen responses predicted assayed lactase activity in all patients with isolated lactase insufficiency, but were "falsely negative" in four of ten children whose lactase insufficiency was secondary to mucosal injury. In both clinical groups, lactose malabsorbers report significantly more symptoms than absorbers (P less than .001), but neither symptom reports nor tolerance tests are accurate methods for distinguishing lactose malabsorbers from absorbers. Although the lactose breath hydrogen test provides objective documentation of lactose malabsorption, it is equally predictive of assayed lactase activity in all clinical groups.
Heuschkel, R; MacDonald, T; Monteleone, G; Bajaj-Elliott, M; Smith, J; Pender, S
BACKGROUND—Degradation of the extracellular matrix and ulceration of the mucosa are major features of inflammatory bowel disease (IBD). One of the most important enzymes in degrading the matrix and produced in excess by cytokine activated stromal cells, is stromelysin-1. The activity of stromelysin-1 is controlled by tissue inhibitor of metalloproteinase (TIMP-1), its natural inhibitor. In model systems excess stromelysin-1 produces mucosal degradation. METHODS—Quantitative competitive RT-PCR was used to analyse stromelysin-1 and TIMP-1 transcripts; western blotting was used to measure the amount of stromelysin-1 and TIMP-1 protein in biopsy samples from children with IBD. RESULTS—In biopsies from patients with active Crohn's disease (n=24), ulcerative colitis (n=23), and controls (n=16), TIMP-1 transcripts and protein were abundant and unchanged. Stromelysin-1 transcripts and protein were markedly elevated in mucosal biopsies obtained from inflamed sites of patients with active IBD but were not elevated in adjacent endoscopically normal mucosa (n=10). Elevated levels of stromelysin-1 transcripts in active Crohn's disease (n=5) returned to normal levels following treatment with enteral nutrition. CONCLUSIONS—Stromelysin-1 is markedly overexpressed at inflamed sites in patients with IBD whereas TIMP-1 remains unaltered. Excess stromelysin-1 is likely to be responsible for loss of mucosal integrity in IBD. Keywords: inflammatory bowel disease; enteral nutrition; intestine; matrix metalloproteinase; tissue inhibitor of metalloproteinase PMID:10861265
Plank, Lukáš; Balhárek, Tomáš; Szépe, Peter
The authors present a retrospective analysis of follicular lymphomas (FL) and mantle cell lymphomas (MCL) diagnosed according to the WHO classification (2008) in consecutive biopsies of GI organs in a period of 11 years. The series includes 18 patients with FL verified in 22 biopsies and 44 patients with MCL diagnosed in 54 biopsies. FL represented always a solitary tumor, most often - up to ¾ of all the cases - of a small intestine, more often in its jejunoileal than duodenal parts. The biopsies were obtained almost equally by endoscopical approach, they were usually mucosal and rarely polypectomic, as well as by surgical resections (54,5 % and 45,5 % of the cases respectively). FL of grade 3 was identified in approximately 11 % of the cases, while majority of the patients showed FL of grade 1 or 2. Only 2 patients with duodenal FL relapsed and bioptically verified recidives did not show signs of a high grade transformation. Although it was difficult to identify a nodular growth pattern in more common small biopsies, a typical histomorphology and phenotype mostly allowed the FL diagnosis in the majority of the cases. The FL diagnosis had to be supported by detection of BCL2 translocation only in one case. MCL appeared most often in the stomach and large intestine, the small intestinal cases represented less than 23 %. In ¼ of the patients the lymphoma was multifocal and manifested as lymphomatoid polyposis affecting most often both large and small intestine. In a majority of the MCL patients, the diagnosis was done in mucosal and polypectomic endoscopic biopsies, surgical intervention and resection was recorded in less than 10 % of the cases. Most of the patients showed conventional "centrocytic" MCL morphology and approximately ¼ of the cases showed blastoid MCL. The rebiopsies of 9 patients revealed a relaps of the disease which was locoidentical in 5 of them; in other 4 patients the biopsies documented a dissemination to other GI organs. The blastic
Kakkar, Aarti; Wasan, Sharmeel K.
The goal of medical treatment for Crohn's disease includes improving patients' quality of life while reducing the need for hospitalization and surgery. The current medical armamentarium includes 5-aminosalicylates, corticosteroids, immunomodulators, and biologic agents. In the past, response to treatment was measured by clinical improvement in symptoms; however, with the advent of disease-modifying medications, mucosal healing has emerged as an increasingly important goal of therapy. Mucosal healing, or endoscopic remission, is associated with increased rates of clinical remission, fewer hospitalizations, and fewer abdominal surgeries. Both the immunomodulator and biologic classes of medications are effective at inducing mucosal healing. Despite several limitations, mucosal healing has become a desirable and valid measure of disease activity. PMID:21869869
Colombo, Bruno M; Scalvenzi, Thibault; Benlamara, Sarah; Pollet, Nicolas
We know that animals live in a world dominated by bacteria. In the last 20 years, we have learned that microbes are essential regulators of mucosal immunity. Bacteria, archeas, and viruses influence different aspects of mucosal development and function. Yet, the literature mainly covers findings obtained in mammals. In this review, we focus on two major themes that emerge from the comparative analysis of mammals and amphibians. These themes concern: (i) the structure and functions of lymphoid organs and immune cells in amphibians, with a focus on the gut mucosal immune system; and (ii) the characteristics of the amphibian microbiota and its influence on mucosal immunity. Lastly, we propose to use Xenopus tadpoles as an alternative small-animal model to improve the fundamental knowledge on immunological functions of gut microbiota.
Gandi, Padma; Anumala, Naveen; Reddy, Amarender; Chandra, Rampalli Viswa
Mucosal fenestration is an opening or an interstice through the oral mucosa. A lesion which occurs with greater frequency than generally realised, its occurrence is attributed to a myriad of causes. Mucogingival procedures including connective tissue grafts, free gingival grafts and lateral pedicle grafts are generally considered to be the treatment of choice in the closure of a mucosal fenestration. More often, these procedures are performed in conjunction with other procedures such as periradicular surgery and with bone grafts. However, the concomitant use of gingival grafts and bone grafts in mucosal fenestrations secondary to infections in sites exhibiting severe bone loss is highly debatable. In this article, we report two cases of mucosal fenestrations secondary to trauma and their management by regenerative periodontal surgery with the placement of guided tissue regeneration membrane and bone graft. The final outcome was a complete closure of the fenestration in both the cases.
Colombo, Bruno M.; Scalvenzi, Thibault; Benlamara, Sarah; Pollet, Nicolas
We know that animals live in a world dominated by bacteria. In the last 20 years, we have learned that microbes are essential regulators of mucosal immunity. Bacteria, archeas, and viruses influence different aspects of mucosal development and function. Yet, the literature mainly covers findings obtained in mammals. In this review, we focus on two major themes that emerge from the comparative analysis of mammals and amphibians. These themes concern: (i) the structure and functions of lymphoid organs and immune cells in amphibians, with a focus on the gut mucosal immune system; and (ii) the characteristics of the amphibian microbiota and its influence on mucosal immunity. Lastly, we propose to use Xenopus tadpoles as an alternative small-animal model to improve the fundamental knowledge on immunological functions of gut microbiota. PMID:25821449
Reljic, Rajko; Sibley, Laura; Huang, Jen-Min; Pepponi, Ilaria; Hoppe, Andreas; Hong, Huynh A.
Needle-free, mucosal immunization is a highly desirable strategy for vaccination against many pathogens, especially those entering through the respiratory mucosa, such as Mycobacterium tuberculosis. Unfortunately, mucosal vaccination against tuberculosis (TB) is impeded by a lack of suitable adjuvants and/or delivery platforms that could induce a protective immune response in humans. Here, we report on a novel biotechnological approach for mucosal vaccination against TB that overcomes some of the current limitations. This is achieved by coating protective TB antigens onto the surface of inert bacterial spores, which are then delivered to the respiratory tract. Our data showed that mice immunized nasally with coated spores developed humoral and cellular immune responses and multifunctional T cells and, most importantly, presented significantly reduced bacterial loads in their lungs and spleens following pathogenic challenge. We conclude that this new vaccine delivery platform merits further development as a mucosal vaccine for TB and possibly also other respiratory pathogens. PMID:23959722
... taking, including blood thinners such as aspirin and herbal supplements, and whether you have any allergies – especially to ... doctor all medications that you are taking, including herbal supplements, and if you have any allergies, especially to ...
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Apoptotic depletion of infiltrating mucosal lymphocytes associated with Fas ligand expression by Helicobacter pylori-infected gastric mucosal epithelium: human glandular stomach as a site of immune privilege.
H. pylori infection almost invariably results in chronic gastritis, but only a proportion of patients develops severe destruction of epithelial glandular structure or peptic ulcer. To confirm the recent data obtained in testis and eye, showing that Fas ligand is involved in the phenomenon of "immune privilege," expression of Fas receptor and its ligand of the stomach was investigated in a panel of gastric biopsies obtained from patients H. pylori-positive (N = 42) and with H. pylori-negative (N = 18) by two-color flow cytometry. The results show that membrane-bound Fas ligand protein is constitutively expressed on freshly isolated human gastric mucosal epithelium coupled with infiltrating lymphocytes. There was significant overexpression of Fas receptor and its ligand, and a higher frequency of apoptotic cell death detected by TUNEL in epithelium and infiltrating lymphocytes in H. pylori-infected patients. These findings suggest that involvement of Fas receptor and its ligand system contributes to some extent to mucosal damage in H. pylori-associated gastritis. However, the more specific findings are apoptotic depletion of invading mucosal lymphocytes associated with Fas ligand expression by gastric epithelium. These provide the first direct quantitative evidence to support Fas receptor counterattack and/or paracrine fratricide as a mechanism of immune privilege in vivo in the H. pylori-infected glandular stomach.
Labgaa, Ismail; Villanueva, Augusto
Liver cancer has become the second cause of cancer-related death worldwide. Most patients are still diagnosed at intermediate or advanced stage, where potentially curative treatment options are not recommended. Unlike other solid tumors, there are no validated oncogenic addiction loops and the only systemic agent to improve survival in advanced disease is sorafenib. All phase 3 clinical trials testing molecular therapies after sorafenib have been negative, none of which selected patients based on predictive biomarkers of response. Theoretically, analysis of circulating cancer byproducts (e.g., circulating tumor cells, cell-free nucleic acids), namely "liquid biopsy," could provide easy access to molecular tumor information, improve patients' stratification and allow to assess tumor dynamics over time. Recent technical developments and preliminary data from other malignancies indicate that liquid biopsy might have a role in the future management of cancer patients.
... Exams and Tests for Cancer Testing Biopsy and Cytology Specimens for Cancer Waiting to hear a possible ... best decisions about your treatment. Testing Biopsy and Cytology Specimens for Cancer How is cancer diagnosed? Types ...
Timmons, Jennifer; Chang, Elizabeth T.; Wang, Jian-Ying; Rao, Jaladanki N.
The epithelium of gastrointestinal (GI) mucosa has the most rapid turnover rate of any tissue in the body and its integrity is preserved through the dynamic balance between cell migration, proliferation, growth arrest and apoptosis. To maintain tissue homeostasis of the GI mucosa, the rates of epithelial cell division and apoptosis must be highly regulated by various extracellular and intracellular factors including cellular polyamines. Natural polyamines spermidine, spermine and their precursor putrescine, are organic cations in eukaryotic cells and are implicated in the control of multiple signaling pathways and distinct cellular functions. Normal intestinal epithelial growth depends on the available supply of polyamines to the dividing cells in the crypts, and polyamines also regulate intestinal epithelial cell (IEC) apoptosis. Although the specific molecular processes controlled by polyamines remains to be fully defined, increasing evidence indicates that polyamines regulate intestinal epithelial integrity by modulating the expression of various growth-related genes. In this review, we will extrapolate the current state of scientific knowledge regarding the roles of polyamines in gut mucosal homeostasis and highlight progress in cellular and molecular mechanisms of polyamines and their potential clinical applications. PMID:25237589
Mueller, Mattea; Moos, Verena; Heller, Frank; Meyer, Thomas F.; Loddenkemper, Christoph; Bojarski, Christian; Fehlings, Michael; Doerner, Thomas; Allers, Kristina; Aebischer, Toni; Ignatius, Ralf; Schneider, Thomas
The mucosal immune system is relevant for homeostasis, immunity, and also pathological conditions in the gastrointestinal tract. Inducible NO synthase (iNOS)–dependent production of NO is one of the factors linked to both antimicrobial immunity and pathological conditions. Upregulation of iNOS has been observed in human Helicobacter pylori infection, but the cellular sources of iNOS are ill defined. Key differences in regulation of iNOS expression impair the translation from mouse models to human medicine. To characterize mucosal iNOS-producing leukocytes, biopsy specimens from H. pylori–infected patients, controls, and participants of a vaccination trial were analyzed by immunohistochemistry, along with flow cytometric analyses of lymphocytes for iNOS expression and activity. We newly identified mucosal IgA-producing plasma cells (PCs) as one major iNOS+ cell population in H. pylori–infected patients and confirmed intracellular NO production. Because we did not detect iNOS+ PCs in three distinct infectious diseases, this is not a general feature of mucosal PCs under conditions of infection. Furthermore, numbers of mucosal iNOS+ PCs were elevated in individuals who had cleared experimental H. pylori infection compared with those who had not. Thus, IgA+ PCs expressing iNOS are described for the first time, to our knowledge, in humans. iNOS+ PCs are induced in the course of human H. pylori infection, and their abundance seems to correlate with the clinical course of the infection. PMID:27456483
Lung cancer is the leading cause of cancer-related deaths worldwide. Recent implementation of low-dose computed tomography (LDCT) screening is predicted to lead to diagnosis of lung cancer at an earlier stage, with survival benefit. However, there is still a pressing need for biomarkers that will identify individuals eligible for screening, as well as improve the diagnostic accuracy of LDCT. In addition, biomarkers for prognostic stratification of patients with early stage disease, and those that can be used as surrogates to monitor tumor evolution, will greatly improve clinical management. Molecular alterations found in the DNA of tumor cells, such as mutations, translocations and methylation, are reflected in DNA that is released from the tumor into the bloodstream. Thus, in recent years, circulating tumor DNA (ctDNA) has gained increasing attention as a noninvasive alternative to tissue biopsies and potential surrogate for the entire tumor genome. Activating gene mutations found in ctDNA have been proven effective in predicting response to targeted therapy. Analysis of ctDNA is also a valuable tool for longitudinal follow-up of cancer patients that does not require serial biopsies and may anticipate the acquisition of resistance. DNA methylation has also emerged as a promising marker for early detection, prognosis and real-time follow-up of tumor dynamics that is independent of the genomic composition of the primary tumor. This review summarizes the various investigational applications of methylated ctDNA in lung cancer reported to date. It also provides a brief overview of the technologies for analysis of DNA methylation in liquid biopsies, and the challenges that befall the implementation of methylated ctDNA into routine clinical practice. PMID:27826530
Bødker, Julie Støve; Christensen, Heidi Søgaard; Johansen, Preben; Nielsen, Søren; Christiansen, Ilse; Bergmann, Olav Jonas; Bøgsted, Martin; Dybkær, Karen; Vyberg, Mogens; Johnsen, Hans Erik
Background Toxicity of the oral and gastrointestinal mucosa induced by high-dose melphalan is a clinical challenge with no documented prophylactic interventions or predictive tests. The aim of this study was to describe molecular changes in human oral mucosa and to identify biomarkers correlated with the grade of clinical mucositis. Methods and Findings Ten patients with multiple myeloma (MM) were included. For each patient, we acquired three buccal biopsies, one before, one at 2 days, and one at 20 days after high-dose melphalan administration. We also acquired buccal biopsies from 10 healthy individuals that served as controls. We analyzed the biopsies for global gene expression and performed an immunohistochemical analysis to determine HLA-DRB5 expression. We evaluated associations between clinical mucositis and gene expression profiles. Compared to gene expression levels before and 20 days after therapy, at two days after melphalan treatment, we found gene regulation in the p53 and TNF pathways (MDM2, INPPD5, TIGAR), which favored anti-apoptotic defense, and upregulation of immunoregulatory genes (TREM2, LAMP3) in mucosal dendritic cells. This upregulation was independent of clinical mucositis. HLA-DRB1 and HLA-DRB5 (surface receptors on dendritic cells) were expressed at low levels in all patients with MM, in the subgroup of patients with ulcerative mucositis (UM), and in controls; in contrast, the subgroup with low-grade mucositis (NM) displayed 5–6 fold increases in HLA-DRB1 and HLA-DRB5 expression in the first two biopsies, independent of melphalan treatment. Moreover, different splice variants of HLA-DRB1 were expressed in the UM and NM subgroups. Conclusions Our results revealed that, among patients with MM, immunoregulatory genes and genes involved in defense against apoptosis were affected immediately after melphalan administration, independent of the presence of clinical mucositis. Furthermore, our results suggested that the expression levels of HLA
Histopathological confirmation of similar intramucosal distribution of fluorescein in both intravenous administration and local mucosal application for probe-based confocal laser endomicroscopy of the normal stomach
Nonaka, Kouichi; Ohata, Ken; Ban, Shinichi; Ichihara, Shin; Takasugi, Rumi; Minato, Yohei; Tashima, Tomoaki; Matsuyama, Yasushi; Takita, Maiko; Matsuhashi, Nobuyuki; Neumann, Helmut
Probe-based confocal laser endomicroscopy (pCLE) is capable of acquiring in vivo magnified cross-section images of the gastric mucosa. Intravenous injection of fluorescein sodium is used for confocal imaging. However, it is still under debate if local administration of the dye to the mucosa is also effective for confocal imaging as it is not yet clear if topical application also reveals the intramucosal distribution of fluorescein. The objective of this study was to evaluate the intramucosal distribution of fluorescein sodium after topical application and to compare the distribution to the conventional intravenous injection used for confocal imaging. pCLE of the stomach uninfected with Helicobacter pylori was performed in a healthy male employing intravenous administration and local mucosal application of fluorescein. The mucosa of the lower gastric body was biopsied 1 min and 5 min after intravenous administration or local mucosal application of fluorescein, and the distribution of fluorescein in the biopsy samples was examined histologically. Green fluorescence was already observed in the cytoplasm of fundic glandular cells in the biopsied deep mucosa 1 min after local mucosal application of fluorescein. It was also observed in the foveolar lumen and inter-foveolar lamina propria, although it was noted at only a few sites. In the tissue biopsied 5 min after the local mucosal application of fluorescein, green fluorescence was more frequently noted in the cytoplasm of fundic glandular cells than in that 1 min after the local mucosal application of fluorescein, although obvious green fluorescence was not identified in the foveolar lumen or inter-foveolar lamina propria. The distribution of intravenously administered fluorescein in the cytoplasm of fundic glandular cells was also clearly observed similarly to that after local mucosal application of fluorescein. Green fluorescence in more cells was observed in many cells 5 min after intravenous administration compared
Moyes, David L.; Naglik, Julian R.
Interactions between mucosal surfaces and microbial microbiota are key to host defense, health, and disease. These surfaces are exposed to high numbers of microbes and must be capable of distinguishing between those that are beneficial or avirulent and those that will invade and cause disease. Our understanding of the mechanisms involved in these discriminatory processes has recently begun to expand as new studies bring to light the importance of epithelial cells and novel immune cell subsets such as Th17 T cells in these processes. Elucidating how these mechanisms function will improve our understanding of many diverse diseases and improve our ability to treat patients suffering from these conditions. In our voyage to discover these mechanisms, mucosal interactions with opportunistic commensal organisms such as the fungus Candida albicans provide insights that are invaluable. Here, we review current knowledge of the interactions between C. albicans and epithelial surfaces and how this may shape our understanding of microbial-mucosal interactions. PMID:21776285
Southern, Peter; Horbul, Julie; Maher, Diane; Davis, Dana A.
Background Candida albicans is a low level commensal organism in normal human populations with the continuous potential to expand and cause a spectrum of clinical conditions. Methodology/Principal Findings Using ex vivo human organ cultures and populations of primary human cells, we have developed several related experimental systems to examine early-stage interactions between C. albicans and mucosal surfaces. Experiments have been conducted both with exogenously added C. albicans and with overtly normal human mucosal surfaces supporting pre-existing infections with natural isolates of Candida. Under different culture conditions, we have demonstrated the formation of C. albicans colonies on human target cells and filament formation, equivalent to tissue invasion. Conclusions/Significance These organ culture systems provide a valuable new resource to examine the molecular and cellular basis for Candida colonization of human mucosal surfaces. PMID:18446191
Agarwal, S. K.; Sethi, S.; Dinda, A. K.
The introduction of the kidney biopsy is one of the major events in the history of nephrology. Primary indications of kidney biopsy are glomerular hematuria/proteinuria with or without renal dysfunction and unexplained renal failure. Kidney biopsy is usually performed in prone position but in certain situations, supine and lateral positions may be required. Biopsy needles have changed with times from Vim–Silverman needle to Tru-cut needle to spring-loaded automatic gun. The procedure has also changed from blind bedside kidney biopsy to ultrasound marking to real-time ultrasound guidance to rarely computerized tomography guidance and laparoscopic and open biopsy. In very specific situations, transjugular kidney biopsy may be required. Most of the centers do kidney biopsy on short 1-day admission, whereas some take it as an outdoor procedure. For critical interpretation of kidney biopsy, adequate sample and clinical information are mandatory. Tissue needs to be stained with multiple stains for delineation of various components of kidney tissue. Many consider that electron microscopy (EM) is a must for all kidney biopsies, but facilities for EM are limited even in big centers. Sophisticated tests such as immunohistochemistry and in-situ hybridization are useful adjuncts for definitive diagnosis in certain situations. PMID:23960337
Daferera, Niki; Kumawat, Ashok Kumar; Hultgren-Hörnquist, Elisabeth; Ignatova, Simone; Ström, Magnus; Münch, Andreas
In this case report, we examined the levels of cytokines expressed before and during fecal stream diversion and after intestinal continuity was restored in a patient with collagenous colitis. We report the case of a 46-year-old woman with chronic, active collagenous colitis who either failed to achieve clinical remission or experienced adverse effects with the following drugs: loperamide, cholestyramine, budesonide, methotrexate and adalimumab. Due to the intractable nature of the disease and because the patient was having up to 15 watery bowel movements per day, she underwent a temporary ileostomy. Colonic biopsies were analyzed for mucosal cytokine protein levels before and during fecal stream diversion and after intestinal continuity was restored. Mucosal protein levels of interleukin (IL)-1β, IL-2, IL-6, IL-12, IL-17 A, IL-23, TNF, IFN-γ, IL-4, IL-5, IL-10 and IL-13 were all higher during active disease and decreased to non-detectable or considerably lower levels during fecal stream diversion. One month after the restoration of bowel continuity, when the patient experienced a relapse of symptoms, IL-2, IL-23 and IL-21 levels were again increased. Our results indicate that fecal stream diversion in this patient suppressed the levels of all cytokines analyzed in colonic biopsies. With the recurrence of clinical symptoms and histological changes after bowel reconstruction, the levels of primarily proinflammatory cytokines increased. Our findings support the hypothesis that a luminal factor triggers the inflammation observed in collagenous colitis. PMID:26019474
Sumagin, R; Brazil, JC; Nava, P; Nishio, H; Alam, A; Luissint, AC; Weber, DA; Neish, AS; Nusrat, A; Parkos, CA
A characteristic feature of gastrointestinal tract inflammatory disorders, such as inflammatory bowel disease, is polymorphonuclear neutrophil (PMN) transepithelial migration (TEM) and accumulation in the gut lumen. PMN accumulation within the intestinal mucosa contributes to tissue injury. While epithelial infiltration by large numbers of PMNs results in mucosal injury, we found that PMN interactions with luminal epithelial membrane receptors may also play a role in wound healing. Intercellular adhesion molecule-1 (ICAM-1) is a PMN ligand that is upregulated on apical surfaces of intestinal epithelial cells under inflammatory conditions. In our study, increased expression of ICAM-1 resulted in enhanced PMN binding to the apical epithelium, which was associated with reduced PMN apoptosis. Following TEM, PMN adhesion to ICAM-1 resulted in activation of Akt and β-catenin signaling, increased epithelial-cell proliferation, and wound healing. Such responses were ICAM-1 dependent as engagement of epithelial ICAM-1 by antibody-mediated cross-linking yielded similar results. Furthermore, using an in-vivo biopsy-based, colonic-mucosal-injury model, we demonstrated epithelial ICAM-1 plays an important role in activation of epithelial Akt and β-catenin signaling and wound healing. These findings suggest that post-migrated PMNs within the intestinal lumen can regulate epithelial homeostasis, thereby identifying ICAM-1 as a potential therapeutic target for promoting mucosal wound healing. PMID:26732677
Sumagin, R; Brazil, J C; Nava, P; Nishio, H; Alam, A; Luissint, A C; Weber, D A; Neish, A S; Nusrat, A; Parkos, C A
A characteristic feature of gastrointestinal tract inflammatory disorders, such as inflammatory bowel disease, is polymorphonuclear neutrophil (PMN) transepithelial migration (TEM) and accumulation in the gut lumen. PMN accumulation within the intestinal mucosa contributes to tissue injury. Although epithelial infiltration by large numbers of PMNs results in mucosal injury, we found that PMN interactions with luminal epithelial membrane receptors may also play a role in wound healing. Intercellular adhesion molecule-1 (ICAM-1) is a PMN ligand that is upregulated on apical surfaces of intestinal epithelial cells under inflammatory conditions. In our study, increased expression of ICAM-1 resulted in enhanced PMN binding to the apical epithelium, which was associated with reduced PMN apoptosis. Following TEM, PMN adhesion to ICAM-1 resulted in activation of Akt and β-catenin signaling, increased epithelial-cell proliferation, and wound healing. Such responses were ICAM-1 dependent as engagement of epithelial ICAM-1 by antibody-mediated cross-linking yielded similar results. Furthermore, using an in-vivo biopsy-based, colonic-mucosal-injury model, we demonstrated epithelial ICAM-1 has an important role in activation of epithelial Akt and β-catenin signaling and wound healing. These findings suggest that post-migrated PMNs within the intestinal lumen can regulate epithelial homeostasis, thereby identifying ICAM-1 as a potential therapeutic target for promoting mucosal wound healing.
van Unen, Vincent; Li, Na; Molendijk, Ilse; Temurhan, Mine; Höllt, Thomas; van der Meulen-de Jong, Andrea E; Verspaget, Hein W; Mearin, M Luisa; Mulder, Chris J; van Bergen, Jeroen; Lelieveldt, Boudewijn P F; Koning, Frits
Inflammatory intestinal diseases are characterized by abnormal immune responses and affect distinct locations of the gastrointestinal tract. Although the role of several immune subsets in driving intestinal pathology has been studied, a system-wide approach that simultaneously interrogates all major lineages on a single-cell basis is lacking. We used high-dimensional mass cytometry to generate a system-wide view of the human mucosal immune system in health and disease. We distinguished 142 immune subsets and through computational applications found distinct immune subsets in peripheral blood mononuclear cells and intestinal biopsies that distinguished patients from controls. In addition, mucosal lymphoid malignancies were readily detected as well as precursors from which these likely derived. These findings indicate that an integrated high-dimensional analysis of the entire immune system can identify immune subsets associated with the pathogenesis of complex intestinal disorders. This might have implications for diagnostic procedures, immune-monitoring, and treatment of intestinal diseases and mucosal malignancies.
Stoopler, Eric T; Sollecito, Thomas P
Oral mucosal diseases encompass several common conditions that affect the general population. Some of these disorders present with signs and symptoms that are pathognomonic for the condition, whereas others present with similar features that can make clinical diagnosis difficult to achieve. It is important for physicians to have a clear understanding of these disorders to provide appropriate care to patients. This article reviews clinical aspects of common oral mucosal disorders, including candidiasis, herpes simplex viral infections, aphthous stomatitis, lichen planus, pemphigus vulgaris, and mucous membrane pemphigoid.
Lu, Yingying; Chen, Jing; Zheng, Junyuan; Hu, Guoyong; Wang, Jingjing; Huang, Chunlan; Lou, Lihong; Wang, Xingpeng; Zeng, Yue
Recent reports have suggested that the gut microbiota is involved in the progression of colorectal cancer (CRC). The composition of gut microbiota in CRC precursors has not been adequately described. To characterize the structure of adherent microbiota in this disease, we conducted pyrosequencing-based analysis of 16S rRNA genes to determine the bacterial profile of normal colons (healthy controls) and colorectal adenomas (CRC precursors). Adenoma mucosal biopsy samples and adjacent normal colonic mucosa from 31 patients with adenomas and 20 healthy volunteers were profiled using the Illumina MiSeq platform. Principal coordinate analysis (PCoA) showed structural segregation between colorectal adenomatous tissue and control tissue. Alpha diversity estimations revealed higher microbiota diversity in samples from patients with adenomas. Taxonomic analysis illustrated that abundance of eight phyla (Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Chloroflexi, Cyanobacteria, Candidate-division TM7, and Tenericutes) was significantly different. In addition, Lactococcus and Pseudomonas were enriched in preneoplastic tissue, whereas Enterococcus, Bacillus, and Solibacillus were reduced. However, both PCoA and cluster tree analyses showed similar microbiota structure between adenomatous and adjacent non-adenoma tissues. These present findings provide preliminary experimental evidence supporting that colorectal preneoplastic lesion may be the most important factor leading to alterations in bacterial community composition. PMID:27194068
Sharma, Karun V.; Venkatesan, Aradhana M.; Swerdlow, Daniel; DaSilva, Daniel; Beck, Avi; Jain, Nidhi; Wood, Bradford J.
Image-guided biopsy is a safe and well-established technique that is familiar to most interventional radiologists (IRs). Improvements in image-guidance, biopsy tools and biopsy techniques now routinely allow for safe biopsy of renal and adrenal lesions which traditionally were considered difficult to reach or technically challenging. Image-guided biopsy is used to establish the definitive tissue diagnosis in adrenal mass lesions that can not be fully characterized with imaging or laboratory tests alone. It is also used to establish definitive diagnosis in some cases of renal parenchymal disease and has an expanding role in diagnosis and characterization of renal masses prior to treatment. Although basic principles and techniques for image-guided needle biopsy are similar regardless of organ, this paper will highlight some technical considerations, indications and complications which are unique to the adrenal gland and kidney because of their anatomic location and physiologic features. PMID:20540919
Trindade, Leda Maria Delmondes Freitas; Menezes, Lania Barreto de Oliveira; de Souza Neta, Adozina Marques; Leite Rolemberg, Paulo Candido; Souza, Lais Dantas; Barreto, Ikaro Daniel de Carvalho; Meurer, Luise
Background Helicobacter pylori (H. pylori) infection affects about 50% of the world population and its association with environmental factors and host properties is involved in gastric carcinogenesis. The study aimed to estimate the prevalence of H. pylori in samples of gastric mucosa biopsies, correlate the presence of the bacteria in the sample with the variables age, sex and origin, to identify the types of lesions found in patients with H. pylori, and to evaluate the association of the lesions with the region of the gastric mucosa. Methods A cross-sectional, retrospective study was carried out in Aracaju, Sergipe, Brazil, from January 2013 to December 2015. A total of 45,206 gastric mucosal biopsies were obtained from patients submitted to upper gastrointestinal endoscopy. Of the reports evaluated, 12,909 met the inclusion criteria since they presented the patient’s demographic data as well as the histopathological characteristics of gastric mucosal regions and positivity for H. pylori. Data were analyzed by IBM SPSS Statistic 20 and subjected to descriptive analyses (categorical variables) and inferential (Pearson’s Qui-square and linear association tests) and multiple correspondence analyses. Significance level adopted 5%. Results Of the total of 12,909 (28.6%) reports evaluated, 67% (8,647) came from urban areas and 64.5% (8,320) were female. The mean age (standard deviation (SD)) was 43 years, ranging from 8 to 100 years, prevailing between 21 and 60 years. Among the types of gastric mucosa analyzed, 95.5% (12,322) were of the antral mucosa. The absence of glandular atrophy, the mild infection intensity for H. pylori, the absence of metaplasia, the presence of foveolar hyperplasia and lymphoid follicles were statistically significant (P < 0.001) in this region. In the fundic region, the evidence of fibrinoleucocytic crust and lymphoid follicles was significant (P < 0.001). There was no evidence of associated ulcerated lesions or significant relationship
Xu, Guan; Tomlins, Scott A.; Siddiqui, Javed; Davis, Mandy A.; Kunju, Lakshmi P.; Wei, John T.; Wang, Xueding
Photoacoustic (PA) measurements encode the information associated with both physical microstructures and chemical contents in biological tissues. A two-dimensional physio-chemical spectrogram (PCS) can be formulated by combining the power spectra of PA signals acquired at a series of optical wavelengths. The analysis of PCS, or namely PA physio-chemical analysis (PAPCA), enables the quantification of the concentrations and the spatial distributions of a variety of chemical components in the tissue. The chemical components and their distribution are the two major features observed in the biopsy procedures which have been regarded as the gold standard of the diagnosis of many diseases. Taking non-alcoholic fatty liver disease and prostate cancer for example, this study investigates the feasibility of PAPCA in characterizing the histopathological changes in the diseased conditions in biological tissue. A catheter based setup facilitating measurement in deep tissues was also proposed and tested.
Preiser, Jean-Charles; Peres-Bota, Daliana; Eisendrath, Pierre; Vincent, Jean-Louis; Van Gossum, André
Background Addition of glutamine to enteral nutrition formulas is consistently associated with a significant decrease in septic morbidity in critically ill patients, possibly related to the attenuation of gut dysfunction. This pilot study was undertaken to compare the effects of enteral administration of two glutamine-enriched formulas containing either additional free glutamine or glutamine-rich proteins, with a standard solution on plasma and mucosal concentrations of glutamine in patients admitted in the Department of Intensive Care. Methods Following randomization, glutamine concentration was determined in endoscopically sampled duodenal biopsies and plasma, before and after a 7-day period of continuous administration of the designated solution. Results The mucosal concentration of glutamine increased in the duodenal biopsies sampled from patients randomized to the solution containing the glutamine-rich proteins (from 3.6 ± 2.2 to 6.7 ± 5.2 micro-mol/g protein), but not from the others. There were no differences between the 3 groups in the plasma concentrations of glutamine, which remained stable over time. Conclusion The source of supplemental glutamine can influence gut mucosal glutamine concentrations, suggesting differences in its availability or utilization. PMID:14613506
Piccolomini, R; Di Bonaventura, G; Festi, D; Catamo, G; Laterza, F; Neri, M
The aim of the present study was to compare eight media, four nonselective and four selective media, to determine the best combination of media for the primary isolation of Helicobacter pylori. Over a period of 5 months, mucosal antral biopsy specimens were obtained from 222 consecutive dyspeptic patients undergoing endoscopy. Biopsy samples were plated in parallel on all eight media. Egg yolk emulsion agar (EYE), Skirrow's medium, Dent's medium, and modified Thayer-Martin medium were used as selective media; modified chocolate agar (MCHOC), Triptycase soy agar (TSA), brucella agar, and brain heart infusion agar were used as nonselective media. Overall, by using these eight media, H. pylori was recovered from biopsy specimens from 114 of 222 patients, yielding an isolation rate of 51%. Comparison of all possible combinations of the eight media showed that the highest rate of isolation of H. pylori was 100% (114 of 114) with EYE-MCHOC, followed by 96.5% (110 of 114) when EYE-TSA was used. Conversely, it was found that none of the media used alone yielded a 100% rate of recovery (the maximum recovery rate was 95%, which was achieved with EYE). These results indicate that the association of EYE and MCHOC yielded the maximum recovery of H. pylori from gastric biopsy specimens. Therefore, the use of selective and nonselective media in parallel offers optimal recovery rates with only a slight increase in costs. PMID:9163478
Boaventura, Viviane S; Cafe, Virginia; Costa, Jackson; Oliveira, Fabiano; Bafica, Andre; Rosato, Andrea; de Freitas, Luiz A R; Brodskyn, Claudia; Barral-Netto, Manoel; Barral, Aldina
Mucosal leishmaniasis (ML) is often clinically silent until reaching a highly advanced state. In this prospective study, 6 of 220 patients with early cutaneous leishmaniasis were diagnosed with mucosal involvement by otorhinolaryngological examination (a rate similar to the reported rate of late ML). Detection of early ML may represent an important strategy in preventing severe mucosal destruction in human leishmaniasis.
Ascierto, Paolo Antonio; Accorona, Remo; Botti, Gerardo; Farina, Davide; Fossati, Piero; Gatta, Gemma; Gogas, Helen; Lombardi, Davide; Maroldi, Roberto; Nicolai, Piero; Ravanelli, Marco; Vanella, Vito
Mucosal melanoma of the head and neck is a very rare and aggressive malignancy with a very poor prognosis. The nasal cavity, paranasal sinuses, and oral cavity are the most common locations. One-, 3- and 5-year survival rates between 2000 and 2007 were 63%, 30% and 20%, respectively. Cigarette smoking seems to be a risk factor even though the evidence for this is very low. Clinical signs and symptoms are usually nonspecific. While surgery is considered the mainstay of treatment for most mucosal melanomas of the head and neck region, radiotherapy has a role in local control of the disease after surgery. Many new treatment options in the last years, in particular targeted therapies (i.e. inhibitors of c-KIT, NRAS/MEK or BRAF) and immunotherapies (anti CTLA-4 and anti PD-1/PD-L1 antibodies), have changed the history of cutaneous melanoma. Despite the different biology, mucosal melanoma is currently treated in the same way as cutaneous melanoma; however, patients with mucosal melanoma were excluded from the majority of recent clinical trials. Recent molecular findings offer new hope for the development of more effective systemic therapy.
Dongari-Bagtzoglou, Anna; Kashleva, Helena; Dwivedi, Prabhat; Diaz, Patricia; Vasilakos, John
C. albicans triggers recurrent infections of the alimentary tract mucosa that result from biofilm growth. Although the ability of C. albicans to form a biofilm on abiotic surfaces has been well documented in recent years, no information exists on biofilms that form directly on mucosal surfaces. The objectives of this study were to characterize the structure and composition of Candida biofilms forming on the oral mucosa. We found that oral Candida biofilms consist of yeast, hyphae, and commensal bacteria, with keratin dispersed in the intercellular spaces. Neutrophils migrate through the oral mucosa and form nests within the biofilm mass. The cell wall polysaccharide β-glucan is exposed during mucosal biofilm growth and is more uniformly present on the surface of biofilm organisms invading the oral mucosa. We conclude that C. albicans forms complex mucosal biofilms consisting of both commensal bacterial flora and host components. These discoveries are important since they can prompt a shift of focus for current research in investigating the role of Candida-bacterial interactions in the pathogenesis of mucosal infections as well as the role of β-glucan mediated signaling in the host response. PMID:19956771
Grande, Elisabetta; Ferranti, Silvia; Gaggiano, Carla; Di Virgilio, Nicola; Vascotto, Marina
We report the case of a two-year-five-month-old child who underwent screening for celiac disease due to strong familiarity. During the first observation body weight and height were at 25th and 50th centile for gender and age. Physical examination did not reveal any sign of disease. Blood tests showed increased transaminases levels and antibodies research showed: tTG IgA: 100 UI/ml, tTG IgG: 36,6 UI/ml, EMA IgA: positive. HLA study revealed homozygous allelic combination DRB1*07;DQA102:01; DQB1* 02:02 with presence of a double copy of beta chain in the composition of the DQ2 heterodymer. Biopsy with histological examination did find neither mucosal alteration nor lymphocytic infiltrates (Marsh 0). During follow up with free diet the patient remained asymptomatic and all antibody titers decreased up to normalization. According to ESPGHAN guidelines the finding of hypertransaminasemia as sign of celiac hepatic inflammation, a more than 10-fold increase of tTG IgA and a high-risk HLA would permit diagnosis of celiac disease but histological examination done due to mismatch between paucity of clinical sings and a "multiple risk combination" excluded it, allowing diagnosis of potential celiac disease. We believe that this case is interesting because of its being in contrast with current literature data that suggest a linear relationship between antibodies levels and histological damage with tTG IgA at the upper reference range in case of potential celiac disease. According to guidelines we could have avoided intestinal biopsy but we would have considered as celiac a patient who is maybe just potentially affected.
Hawkey, C J; Hawthorne, A B; Hudson, N; Cole, A T; Mahida, Y R; Daneshmend, T K
1. An increasing body of data suggests that the antihaemostatic as well as the ulcerogenic actions of aspirin and other non-steroidal anti-inflammatory drugs may be operative when patients present with haematemesis and melaena. 2. We therefore developed methods to allow separate evaluation of the erosive and anti-haemostatic actions of aspirin in the human gastric mucosa. Volunteer subjects took 300 mg of aspirin daily in the morning or 600 mg of aspirin four times a day for 5 days under blinded randomized conditions. Changes in spontaneous gastric microbleeding, endoscopic signs of injury, spontaneous bleeding per gastric erosion, biopsy-induced bleeding and eicosanoids were studied. 3. Both doses of aspirin significantly inhibited gastric mucosal synthesis of prostaglandin E2 and reduced the serum thromboxane concentration. Erosions developed and regressed rapidly; compared with baseline 300 mg of aspirin daily in the morning caused substantial numbers of gastric erosions to develop (mean 5.3, 95% confidence limits 2.7-10.2) but this was significantly less than that caused by 600 mg of aspirin four times a day (10.9, 7.2-16.5, P less than 0.05). The presence of erosions was associated with enhanced spontaneous bleeding, but only during aspirin administration. 4. Aspirin significantly increased bleeding induced by mucosal biopsy and was associated with significant enhancements in the rate of bleeding per gastric erosion. Bleeding rate per erosion but not biopsy-induced bleeding showed a significant dose-related increase with 600 mg of aspirin four times a day. Enteric coating reduced endoscopic signs of injury, but did not affect the impaired haemostasis caused by aspirin.(ABSTRACT TRUNCATED AT 250 WORDS)
Quispe Calla, N E; Vicetti Miguel, R D; Boyaka, P N; Hall-Stoodley, L; Kaur, B; Trout, W; Pavelko, S D; Cherpes, T L
Depot-medroxyprogesterone acetate (DMPA) is a hormonal contraceptive especially popular in areas with high prevalence of HIV and other sexually transmitted infections (STI). Although observational studies identify DMPA as an important STI risk factor, mechanisms underlying this connection are undefined. Levonorgestrel (LNG) is another progestin used for hormonal contraception, but its effect on STI susceptibility is much less explored. Using a mouse model of genital herpes simplex virus type 2 (HSV-2) infection, we herein found that DMPA and LNG similarly reduced genital expression of the desmosomal cadherin desmoglein-1α (DSG1α), enhanced access of inflammatory cells to genital tissue by increasing mucosal epithelial permeability, and increased susceptibility to viral infection. Additional studies with uninfected mice revealed that DMPA-mediated increases in mucosal permeability promoted tissue inflammation by facilitating endogenous vaginal microbiota invasion. Conversely, concomitant treatment of mice with DMPA and intravaginal estrogen restored mucosal barrier function and prevented HSV-2 infection. Evaluating ectocervical biopsy tissue from women before and 1 month after initiating DMPA remarkably revealed that inflammation and barrier protection were altered by treatment identically to changes seen in progestin-treated mice. Together, our work reveals DMPA and LNG diminish the genital mucosal barrier; a first-line defense against all STI, but may offer foundation for new contraceptive strategies less compromising of barrier protection.
Calla, Nirk E Quispe; Miguel, Rodolfo D Vicetti; Boyaka, Prosper N; Hall-Stoodley, Luanne; Kaur, Balveen; Trout, Wayne; Pavelko, Stephen D; Cherpes, Thomas L
Depot-medroxyprogesterone acetate (DMPA) is a hormonal contraceptive especially popular in areas with high prevalence of HIV and other sexually transmitted infections (STI). While observational studies identify DMPA as an important STI risk factor, mechanisms underlying this connection are undefined. Levonorgestrel (LNG) is another progestin used for hormonal contraception, but its effect on STI susceptibility is much less explored. Using a mouse model of genital HSV-2 infection, we herein found DMPA and LNG similarly reduced genital expression of the desmosomal cadherin desmoglein-1α (DSG1α), enhanced access of inflammatory cells to genital tissue by increasing mucosal epithelial permeability, and increased susceptibility to viral infection. Additional studies with uninfected mice revealed DMPA-mediated increases in mucosal permeability promoted tissue inflammation by facilitating endogenous vaginal microbiota invasion. Conversely, concomitant treatment of mice with DMPA and intravaginal estrogen restored mucosal barrier function and prevented HSV-2 infection. Evaluating ectocervical biopsy tissue from women before and 1 month after initiating DMPA remarkably revealed inflammation and barrier protection were altered by treatment identically to changes seen in progestin-treated mice. Together, our work reveals DMPA and LNG diminish the genital mucosal barrier; a first-line defense against all STI, but may offer foundation for new contraceptive strategies less compromising of barrier protection. PMID:27007679
Van Sebille, Ysabella Z A; Stansborough, Romany; Wardill, Hannah R; Bateman, Emma; Gibson, Rachel J; Keefe, Dorothy M
Chemotherapy-induced mucositis is a common condition caused by the breakdown of the mucosal barrier. Symptoms can include pain, vomiting and diarrhoea, which can often necessitate chemotherapy treatment breaks or dose reductions, thus compromising survival outcomes. Despite the significant impact of mucositis, there are currently limited clinically effective pharmacological therapies for the pathology. New emerging areas of research have been proposed to play key roles in the development of mucositis, providing rationale for potential new therapeutics for the prevention, treatment or management of chemotherapy-induced mucositis. This review aims to address these new areas of research and to comment on the therapeutics arising from them.
Loran, Oleg B; Veliev, Evgeny I; Sokolov, Egor A; Dadashev, Elmar O; Guspanov, Renat I
Transrectal prostate biopsy is considered a relatively safe procedure, with a quite small number of complications. We report a patient with a rectourethral fistula after a repeat transrectal prostate biopsy. To our knowledge, this is the first incident in the published literature.
Ansari, Junaid; Yun, Jungmi W; Kompelli, Anvesh R; Moufarrej, Youmna E; Alexander, Jonathan S; Herrera, Guillermo A; Shackelford, Rodney E
The incidence of lung cancer has significantly increased over the last century, largely due to smoking, and remains the most common cause of cancer deaths worldwide. This is often due to lung cancer first presenting at late stages and a lack of curative therapeutic options at these later stages. Delayed diagnoses, inadequate tumor sampling, and lung cancer misdiagnoses are also not uncommon due to the limitations of the tissue biopsy. Our better understanding of the tumor microenvironment and the systemic actions of tumors, combined with the recent advent of the liquid biopsy, may allow molecular diagnostics to be done on circulating tumor markers, particularly circulating tumor DNA. Multiple liquid biopsy molecular methods are presently being examined to determine their efficacy as surrogates to the tumor tissue biopsy. This review will focus on new liquid biopsy technologies and how they may assist in lung cancer detection, diagnosis, and treatment.
Ansari, Junaid; Yun, Jungmi W.; Kompelli, Anvesh R.; Moufarrej, Youmna E.; Alexander, Jonathan S.; Herrera, Guillermo A.; Shackelford, Rodney E.
The incidence of lung cancer has significantly increased over the last century, largely due to smoking, and remains the most common cause of cancer deaths worldwide. This is often due to lung cancer first presenting at late stages and a lack of curative therapeutic options at these later stages. Delayed diagnoses, inadequate tumor sampling, and lung cancer misdiagnoses are also not uncommon due to the limitations of the tissue biopsy. Our better understanding of the tumor microenvironment and the systemic actions of tumors, combined with the recent advent of the liquid biopsy, may allow molecular diagnostics to be done on circulating tumor markers, particularly circulating tumor DNA. Multiple liquid biopsy molecular methods are presently being examined to determine their efficacy as surrogates to the tumor tissue biopsy. This review will focus on new liquid biopsy technologies and how they may assist in lung cancer detection, diagnosis, and treatment. PMID:28191282
Schild, H H; Michel, S G
To examine the quality and comparability of biopsy needles. 310 biopsy needles of 40 types were examined microscopically and their suitability for penetrating tissues were studied. For this purpose the pressure required to penetrate a phantom was evaluated. The results were correlated with the diameter, design and construction. 50 of the 310 needles (16%) showed faults on microscopic examination. Measurements of identical needle types within a single production run showed variations in penetration pressures up to 330% (measured as force required for penetration), the average variation was 88%. The results show that the quality of biopsy needles is not constant and leaves something to be desired. To what extent variations in quality affect the biopsy material cannot be estimated. Nevertheless, the present results indicate that stricter quality control of biopsy needles is desirable.
Owen, Jennifer L.; Sahay, Bikash; Mohamadzadeh, Mansour
As most infectious organisms gain entry at mucosal surfaces, there is a great deal of interest in developing vaccines that elicit effective mucosal immune responses against pathogen challenge. Targeted vaccination is one of the most effective methods available to prevent and control infectious diseases. Mucosal vaccines can offer lower costs, better accessibility, needle free delivery, and a higher capacity for mass immunizations during pandemics. Both local mucosal immunity and robust systemic responses can be achieved through mucosal vaccination. Recent progress in understanding the molecular and cellular components of the mucosal immune system have allowed for the development of a novel mucosal vaccine platform utilizing specific dendritic cell-targeting peptides and orally administered lactobacilli to elicit efficient antigen specific immune responses against infections, including B. anthracis in experimental models of disease. PMID:23835515
Owen, Jennifer L; Sahay, Bikash; Mohamadzadeh, Mansour
As most infectious organisms gain entry at mucosal surfaces, there is a great deal of interest in developing vaccines that elicit effective mucosal immune responses against pathogen challenge. Targeted vaccination is one of the most effective methods available to prevent and control infectious diseases. Mucosal vaccines can offer lower costs, better accessibility, needle free delivery, and a higher capacity for mass immunizations during pandemics. Both local mucosal immunity and robust systemic responses can be achieved through mucosal vaccination. Recent progress in understanding the molecular and cellular components of the mucosal immune system have allowed for the development of a novel mucosal vaccine platform utilizing specific dendritic cell-targeting peptides and orally administered lactobacilli to elicit efficient antigen specific immune responses against infections, including Bacillus anthracis in experimental models of disease.
Masthan, Mahaboob Kader; Narayanasamy, Aravindha Babu
Aims and Objectives To determine the association of oral mucosal lesions in a group of Chennai population aged 15 years and above with smoking and chewing habits. To also determine the dose-response relationship of these habits associated with the risk of oral mucosal lesions. Materiala and Methods The study was undertaken with 450 subjects with smoking and/or chewing habits aged 15 years and over gathered through random selection in Chennai, India. Subjects with alcohol intake were excluded from the study. Based on the habits the study group was categorized into smokers, chewers and mixed (smoking+chewing). One hundred and fifty subjects diagnosed with oral mucosal lesions designated as “cases” and 300 lesion-free “controls”, frequency matched for age, sex, habit and family income were assessed during the study. The study protocol included a visual oral soft tissue examination and a questionnaire-based interview. In addition, those requiring further examination, scalpel biopsies were performed to establish a definitive diagnosis. Results Irrespective of the type of habit, 78% of cases smoked and/or chewed for more than 10 years as compared to 37.4% of the control group. Similarly, 71.3% of cases smoked and/or chewed more than 5 times per day as compared to 25.6% of the control group. Eleven habits related mucosal lesions of the oral cavity were encountered. Smoker’s melanosis was the most common oral mucosal lesion followed by Oral submucous fibrosis and Leukoplakia. Dose-response relationships were observed for both duration and frequency of habits on the risk of oral mucosal lesions. Conclusion The result of the present study provides information on the association of oral mucosal lesions in smokers, chewers and patients with mixed habits. The mucosal lesions encountered included a few potentially malignant conditions and oral squamous cell carcinoma. Habits were more prevalent in men thus more lesions were encountered in males than in females
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Gastroenterology-urology biopsy instrument. 876... Gastroenterology-urology biopsy instrument. (a) Identification. A gastroenterology-urology biopsy instrument is a... generic type of device includes the biopsy punch, gastrointestinal mechanical biopsy instrument,...
Matsubara, Victor H; Bandara, H M H N; Mayer, Marcia P A; Samaranayake, Lakshman P
Candidais an opportunistic pathogen that causes mucosal and deep systemic candidiasis. The emergence of drug resistance and the side effects of currently available antifungals have restricted their use as long-term prophylactic agents for candidal infections. Given this scenario, probiotics have been suggested as a useful alternative for the management of candidiasis. We analyzed the available data on the efficacy of probiotics in candidal colonization of host surfaces. A number of well-controlled studies indicate that probiotics, particularly lactobacilli, suppressCandidagrowth and biofilm development in vitro.A few clinical trials have also shown the beneficial effects of probiotics in reducing oral, vaginal, and enteric colonization byCandida; alleviation of clinical signs and symptoms; and, in some cases, reducing the incidence of invasive fungal infection in critically ill patients. Probiotics may serve in the future as a worthy ally in the battle against chronic mucosal candidal infections.
Toner, Liam; Bolton, Damien M
Purpose Urosepsis is the most feared complication of transrectal prostate biopsy. The incidence may be increasing from <1% to 2%–3% in contemporary series. Historically, fluoroquinolones have been effective antibiotic prophylaxis to prevent infective complications but antibiotic resistance is increasing. The increase in antibiotic resistance may contribute to reported increases in urosepsis and hospitalization after transrectal biopsy. This article will review other methods clinicians may employ to reduce the incidence of infective complications after prostate biopsy. Materials and Methods A systematic review of the literature was conducted using literature databases PubMed and Ovid MEDLINE in August 2015 in accordance with the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) criteria. Results Effective strategies to reduce infective complications after transrectal prostate biopsy include augmented prophylaxis with other antibiotics, rectal swab culture directed antibiotic prophylaxis or a transperineal biopsy approach. Needle disinfection, minimizing the number of biopsy needles and rectal disinfectants may also be of use. These methods may be of particular utility in patients with risk factors for developing urosepsis such as recent antibiotic use and overseas travel. Conclusions The scientific literature describes various techniques designed to reduce infective complications caused by prostate biopsy. Clinicians should consider incorporating these novel techniques into their contemporary practice. PMID:26981590
Iho, Sumiko; Maeyama, Jun-ichi; Suzuki, Fumiko
Bacterial DNA comprising palindromic sequences and containing unmethylated CpG is recognized by toll-like receptor 9 of plasmacytoid dendritic cells (pDCs) and induces the production of interferon-α and chemokines, leading to the activation of a Th1 immune response. Therefore, synthetic equivalents of bacterial DNA (CpG oligodeoxynucleotides) have been developed for clinical applications. They are usually phosphorothioated for in vivo use; this approach also leads to adverse effects as reported in mouse models.Mucosal vaccines that induce both mucosal and systemic immunity received substantial attention in recent years. For their development, phosphodiester-linked oligodeoxynucleotides, including the sequence of a palindromic CpG DNA may be advantageous as adjuvants because their target pDCs are present right there, in the mucosa of the vaccination site. In addition, the probability of adverse effects is believed to be low. Here, we review the discovery of such CpG oligodeoxynucleotides and their possible use as mucosal adjuvants. PMID:25751765
Within the last decades a dramatic increase in allergic diseases has been recognized in the Westernized societies, leading to the fact that meanwhile 25-30% of the population is afflicted by allergic disorders. Besides a hereditary disposition, other factors, including a reduced microbial contact early in life or changes in nutrition, might also have influenced this epidemiological development. So far the only causative treatment against type-I allergies is specific immunotherapy. In young and monosensitized patients this treatment is highly efficacious, while there are clear limitations in older or multisensitized patients. Allergy research therefore aims at establishing new and more efficacious treatment strategies in prophylactic as well as therapeutic settings. Our research programs focus on the development of novel allergy vaccines based on the induction of mucosal tolerance. In different mouse models of respiratory allergy mucosal treatment with genetically engineered allergen constructs proved to prevent the development of allergic mono- and multisensitivities. The additional use of mucosal adjuvants seems particularly important to improve therapeutic treatment approaches. Recent studies on the inverse relation of certain parasite infections and the development of allergy prompted us to search for selected parasitic molecules with immunosuppressive properties as potential adjuvant systems for novel allergy vaccines. An overview of our recent studies will be given.
Mustafa, Mayson B; Porter, Stephen R; Smoller, Bruce R; Sitaru, Cassian
A group of autoimmune diseases is characterised by autoantibodies against epithelial adhesion structures and/or tissue-tropic lymphocytes driving inflammatory processes resulting in specific pathology at the mucosal surfaces and the skin. The most frequent site of mucosal involvement in autoimmune diseases is the oral cavity. Broadly, these diseases include conditions affecting the cell-cell adhesion causing intra-epithelial blistering and those where autoantibodies or infiltration lymphocytes cause a loss of cell-matrix adhesion or interface inflammation. Clinically, patients present with blistering, erosions and ulcers that may affect the skin as well as further mucosal surfaces of the eyes, nose and genitalia. While the autoimmune disease may be suspected based on clinical manifestations, demonstration of tissue-bound and circulating autoantibodies, or lymphocytic infiltrates, by various methods including histological examination, direct and indirect immunofluorescence microscopy, immunoblotting and quantitative immunoassay is a prerequisite for definitive diagnosis. Given the frequency of oral involvement and the fact that oral mucosa is the initially affected site in many cases, the informed practitioner should be well acquainted with diagnostic and therapeutic aspects of autoimmune dermatosis with oral involvement. This paper reviews the pathogenesis and clinical presentation of these conditions in the oral cavity with a specific emphasis on their differential diagnosis and current management approaches.
MacEntee, M I; Glick, N; Stolar, E
The numbers of participants over 75 years of age in previous studies of oral health have not been sufficient to permit a full investigation of the influence of age on the mouth. In this study a disproportionate stratified random sample of 255 independent elders was selected from a list of urban voters to provide similar numbers of men and women in three age groups. The subjects were interviewed and examined, and nearly half of them had mucosal disorders. There was a significant (P < 0.05) association between mucosal lesions and the use of dentures and tobacco, whereas stomatitis, denture-related hyperplasia and angular cheilitis in particular were associated significantly with men and with the use of defective dentures. Logistic regression revealed that neither age alone nor the quality of dentures predispose to mucosal lesions, but that the odds of finding stomatitis, denture-related hyperplasia and angular cheilitis in particular increased about three-fold in denture-users, and almost doubled in men.
Coronel-Banda, Mauricio E; Serra-Renom, Jose M; Lorente, Marian; Larrea-Terán, Wendy P
The cutaneous perforators of the facial artery have been well described, but to our knowledge the oral mucosal perforators have not. We studied 10 facial arteries from 10 hemifaces in 5 cadavers. The arteries were injected with latex, and we studied all perforators that extended from the facial artery and headed directly to the oral mucosa. The diameter and length of the facial artery and its mucosal perforators were measured and compared. We found 52 oral mucosal perforators in the 10 facial arteries injected with latex. Their mean (SD) diameter was 0.5 (0.2) mm and the mean (SD) number/facial artery was 5.2 (1.1). Their mean (SD) length was 16.4 (5.3) mm. Most of those to the cheek were localised between the branching-off points of the inferior and superior labial arteries. The facial artery has perforators to the oral mucosa of the cheek, most of them between the points at which the labial arteries emerge.
Ahmad, R; Sorrell, M F; Batra, S K; Dhawan, P; Singh, A B
Inflammatory bowel disease (IBD) is a multifactorial disease. A breach in the mucosal barrier, otherwise known as "leaky gut," is alleged to promote mucosal inflammation by intensifying immune activation. However, interaction between the luminal antigen and mucosal immune system is necessary to maintain mucosal homeostasis. Furthermore, manipulations leading to deregulated gut permeability have resulted in susceptibility in mice to colitis as well as to creating adaptive immunity. These findings implicate a complex but dynamic association between mucosal permeability and immune homeostasis; however, they also emphasize that compromised gut permeability alone may not be sufficient to induce colitis. Emerging evidence further supports the role(s) of proteins associated with the mucosal barrier in epithelial injury and repair: manipulations of associated proteins also modified epithelial differentiation, proliferation, and apoptosis. Taken together, the role of gut permeability and proteins associated in regulating mucosal inflammatory diseases appears to be more complex than previously thought. Herein, we review outcomes from recent mouse models where gut permeability was altered by direct and indirect effects of manipulating mucosal barrier-associated proteins, to highlight the significance of mucosal permeability and the non-barrier-related roles of these proteins in regulating chronic mucosal inflammatory conditions.
Diniz-Freitas, Marco; Torreira-Lorenzo, Juan-Carlos; García-García, Abel; Gándara-Rey, José M.
Objective: To conduct an analysis of the frequency of oral lesions in biopsies over a 14-year period in the Oral Medicine, Oral Surgery and Implantology Unit. Material and Methods: We conducted a retrospective study of biopsies removed from 1995-2009, recording data regarding age, sex, location of the lesions, biopsy types, anatomical and pathological diagnosis and definitive diagnosis. Results: Of the 562 patients studied, the average age was 51.8 years, with a standard deviation of 18.5 (range 5-96). The distribution by sex was 318 (56.6%) women and 244 (43.4%) men. The most common diagnostic category was mucosal pathologies in 37.9% of cases, followed by odontogenic cysts in 27.8%. Malignant tumors accounted for 3.9% of cases, oral squamous cell carcinomas were the most frequent malignancy, appearing in 22 cases. Bisphosphonate- related osteonecrosis of the jaws was the most common injury within the bone lesions group. Conclusion: Following the performance of 647 biopsies on 562 patients, we can say that the most common injury was radicular cysts (appearing in 108 cases), having found statistical differences in relation to the patients’ sex and age. Key words: Frequency, oral pathology, biopsy. PMID:21743423
Tanideh, N; Namazi, F; Andisheh Tadbir, A; Ebrahimi, H; Koohi-Hosseinabadi, O
Oral mucositis is a common and irritating complication of chemotherapy and radiotherapy for malignancies. Current treatments have failed to achieve complete remission of this complication. The St. John's wort plant (Hypericum perforatum) has long been known for its anti-inflammatory and antibacterial effects. The current study was designed to investigate the therapeutic efficacy of the topical and systemic administration of H. perforatum extract on oral mucositis. Oral mucositis was induced in 72 male golden hamsters by administration of 5-fluorouracil (60mg/kg), on days 0, 5, and 10 of the study. The cheek pouch was scratched with a sterile needle on days 1 and 2. On days 12-17, H. perforatum extract topical gel 10%, oral H. perforatum extract (300mg/kg), and gel base groups were treated and then compared with a control group. Weights and blood samples were evaluated, biopsies from buccal lesions were examined histopathologically, and tissue malondialdehyde (MDA) was measured. Both of the H. perforatum extract treatment groups saw a significant relief in oral mucositis compared to the control and base gel groups; the systemic form was superior to the topical form. H. perforatum extract, administered orally or topically, expedited the healing of chemotherapy-induced oral mucositis in hamsters.
... Index A-Z Ultrasound-Guided Fine Needle Aspiration Biopsy of the Thyroid An ultrasound-guided thyroid biopsy ... Thyroid? What is Ultrasound-Guided Fine Needle Aspiration Biopsy of the Thyroid? During a fine needle aspiration ...
Shirian, Sadegh; Oryan, Ahmad; Hatam, Gholam Reza; Daneshbod, Yahya
Cases of human oro-mucosal leishmaniasis are mainly reported in areas where Leishmania (Viannia) braziliensis perpetuates and the damages are mainly located at the cartilaginous nasal septum and frontal portions of the nasal fossa. In Iran, an area free of any L.(V) braziliensis, three Leishmania species are known to perpetuate through distinct (i) blood-feeding sand flies and (ii) rodents or (iii) canidae. Thus while establishing the diagnosis of any human oro-mucosal lesions, three Leishmania species - L. infantum, L. major, and L. tropica - must be considered as potential etiological agents of these damages. With these objectives in mind, features such as localization, extent, severity of oro-mucosal lesions, and duration of symptoms at the time of diagnosis were recorded from 11 patients with respect to the presence or absence of cutaneous lesions in other body parts. The biopsy samples were collected from the oro-mucosal and cutaneous lesions and were processed for further identification of the Leishmania species. The lesions ranged from mucosal nodules without ulceration, nodules with erosion, and shallow to deep ulcerations. Leishmania major was isolated from six (55%) cases showing lesions or scars. The scars were restricted to upper and lower extremities. For the other five patients who did not display any signs of former or active cutaneous leishmaniasis, L. major, L. tropica, and L. infantum were isolated from their lesions. In conclusion L. major, L. infantum, and L. tropica, regardless of common tropism, can be seen in mucosal tissues. However, L. major was the predominant species detected from the lesions in the nasal, gingival, and hard and soft palates, and L. tropica was isolated from the gingival and lower lip lesions. Leishmania infantum was isolated from two severe cases of deep mucosal damage displayed by the epiglottis, cricoarytenoid muscle, and laryngeal mucosa. One important finding was the association of L. major with active or scarred
Liu, Junhua; Bian, Gaorui; Sun, Daming; Zhu, Weiyun; Mao, Shengyong
This study aims to investigate the effect of starter feeding supplementation on colonic mucosal bacterial communities and on mucosal immune homeostasis in pre-weaned lambs. We selected eight pairs of 10-day-old lamb twins. One twin was fed breast milk (M, n = 8), while the other was fed breast milk plus starter (M+S, n = 8). The lambs were sacrificed at 56 days age. Colonic content was collected to determine the pH and the concentrations of volatile fatty acids (VFA) and lactate. The colonic mucosa was harvested to characterize the bacterial communities using Illumina MiSeq sequencing and to determine mRNA expression levels of cytokines and toll-like receptors (TLR) using quantitative real-time PCR. The results show that starter feeding decreased luminal pH and increased the concentrations of acetate, propionate, butyrate, total VFA, and lactate in the colon. The principal coordinate analysis (PCA) and analysis of molecular variance show that starter feeding supplementation significantly affected the colonic mucosal bacterial communities with a higher relative abundance of the dominant taxa unclassified S24-7, Oscillibacter, Prevotella, Parabacteroides, Bifidobacterium, Ruminobacter, and Succinivibrio, and a lower proportion of unclassified Ruminococcaceae, RC9_gut_group, Blautia, Phocaeicola, Phascolarctobacterium, unclassified BS11_gut_group, unclassified family_XIII, and Campylobacter in lambs. Meanwhile, starter feeding decreased mRNA expression of TLR4 and cytokines TNF-α and IFN-γ in colonic tissue. Furthermore, the changes in the colonic mucosal mRNA expression of TLR and cytokines were associated with changes in mucosal bacterial composition. These findings may provide new insights into colonic mucosal bacteria and immune homeostasis in developing lambs. PMID:28382025
Gilkes, C E; Love, S; Hardie, R J; Edwards, R J; Scolding, N J; Rice, C M
Brain biopsy is well established in clinical practice when there is suspicion of CNS malignancy. However, there is little consensus regarding the indications for brain biopsy in non-malignant neurological disease. This is due in no small part to limitations in the available literature pertaining to diagnostic brain biopsies. The published evidence largely comprises small, retrospective, single-centre analyses performed over long time periods, including non-homogeneous patient groups with considerable variation in reported outcomes. Here we present pragmatic guidance for those clinicians considering diagnostic brain biopsy in a patient with non-neoplastic neurological disease and highlight practice points with the aim of maximising the probability of gaining clinically useful information from the procedure.
Pestka, J M; Salzmann, G M; Südkamp, N P; Niemeyer, P
Autologous chondrocyte implantation (ACI) is an established two-step procedure for the treatment of full-thickness cartilage defects of the knee. Cartilage harvest from the affected knee joint represents the first step of this procedure and is essential for further in vitro expansion of autologous chondrocytes. Nevertheless, the cartilage biopsy process itself is underrepresented in the scientific literature and currently there is only a limited amount of data available addressing this process. Biopsy location as well as the technique itself and instruments used for cartilage collection are not well defined and only little standardisation can be found. The article describes the relevant aspects of the biopsy in the context of ACI with regard to the literature available. Follow-up studies to better define and standardise the cartilage biopsy process are thus required.
... miner who died prior to March 31, 1980, an autopsy or biopsy report shall be considered even when the... concerning a miner who died prior to March 31, 1980, shall be accorded the appropriate weight in light of...
... the Breast . top of page What does the equipment look like? The traditional MRI unit is a ... is used for a surgical biopsy. Other sterile equipment involved in this procedure includes syringes, sponges, forceps, ...
Misra, Vatsala; Misra, S P; Dwivedi, Manisha; Singh, P A
To assess the prevalence of gastric giardiasis in gastric biopsies of patients with carcinoma stomach and in patients taking treatment for duodenal ulcer. Gastric biopsy specimens from 54 patients of carcinoma stomach and 100 antral biopsies from patients taking treatment for duodenal ulcer were included in the study. Sections were stained with haematoxylin and eosin, methylene blue and May Grunwald-Giemsa stains and examined for presence of Giardia lamblia trophozoites. Eight out of 54 (14.9%) biopsies of gastric carcinoma patients harboured trophozoites of Giardia lamblia. Associated H. pylori infection was present in all biopsies (8/8; 100%). Atrophy and intestinal metaplasia was present in 62.5% (5/8) and 25% (2/8) cases respectively. Sections from seven out of 35 patients (20%) taking treatment for duodenal ulcer showed presence of G. lamblia. H. pylori infection, gastritis and atrophy were found in 85.7% (6/7), 71.4% (5/7) and 28.6% (2/7) cases respectively. First gastric biopsy in these patients was negative for G. lamblia but 2nd and 3rd biopsies were positive. A careful search for G. lamblia trophozoites should be made while examining the gastric biopsies, especially in patients with carcinoma stomach, intestinal metaplasia, atrophic gastritis and those taking treatment for duodenal ulcer. This may help in indirect diagnosis of clinically unsuspected cases of intestinal giardiasis and may explain persistence of vague upper gastrointestinal tract (UGIT) symptoms despite clearance of H. pylori in patients on anti-ulcer therapy.
Pandey, Abhishek; Dican, Razvan; Beier, Jörn; Keller, Hansjörg
The use of buccal mucosal grafts for the reconstruction of urethral strictures is an established procedure. Because of its robustness, the buccal mucosal graft could also potentially provide an alternative for other indications in reconstructive urology. We report here six consecutive patients who received a buccal mucosal graft for ureteral strictures, glans reconstruction and stoma stenosis. The follow up for all patients ranged from 26 to 50 months. The buccal mucosal graft showed excellent functional results for the ureteral strictures and stenosis from ureterocutaneostomy. For glans reconstructions, the buccal mucosal grafts delivered excellent cosmetic and functional results without causing meatal stenosis. We conclude the buccal mucosal graft can be used in reconstructive surgery beyond the reconstruction of urethral strictures.
Jezek, D; Knezević, N; Kalanj-Bognar, S; Vukelić, Z; Krhen, I
The aim of the study was to investigate the role of a testicular biopsy in the diagnosis and therapy of infertile men with a non-obstructive azoospermia. Overall, 70 testicular biopsies from infertile men were analysed. Samples were obtained by the "open testicular biopsy" method. After dissection, several pieces of the tissue were immediately immersed into the Sperm Prep Medium (Medi-Cult) and fixative (5.5% buffered glutaraldehyde). Tissue samples transported in Sperm Prep Medium were plunged into Sperm Freezing Medium (Medi-Cult) and were stored in liquid nitrogen for potential in vitro fertilization procedures. The tissue was also processed for semithin sections and transmission electron microscopy. Semithin sections from 8 infertile patients demonstrated regular testis structure and fully preserved spermatogenesis (control biopsies). In the remaining 62 cases, spermatogenesis was impaired and a variety of pathological changes could be seen: disorganization and desquamation of spermatogenic cells, spermatid or spermatocyte "stop", spermatogonia only, "Sertoli cells only" or tubular fibrosis. However, in 65% of cases (despite the above mentioned changes of seminiferous epithelium) foci of preserved spermatogenesis could be detected. These cases were classified as "mixed atrophy" of seminiferous tubules. In 63% of infertile patients, a successful extraction of sperm from the biopsy could be performed. In azoospermic patients, histological analysis of testicular biopsy proved to be very useful in terms of diagnosis as well as therapy, i.e. for further in vitro fertilization procedures.
Seldenrijk, C. A.; Meuwissen, S. G.; Schipper, N. W.; Morson, B. C.; Lindeman, J.; Meijer, C. J.
AIMS: To investigate whether counting cells containing immunoglobulin (Ig) subclass in colonic biopsy specimens of patients with chronic inflammatory bowel disease, in addition to conventional histological evaluation, can improve the differentiation of patients with Crohn's disease from those with ulcerative colitis. METHODS: The colonic and rectal biopsy specimens of 40 patients with chronic inflammatory bowel disease, comprising 20 patients with Crohn's disease and 20 with ulcerative colitis, were used and sections were stained specifically for IgA, IgM, and IgG heavy chains using an indirect immune peroxidase method. The immunoglobulin subclass containing cells were counted using an ocular grid counting method in a light microscope. A linear stepwise discriminant analysis was performed on Ig subclass containing cell counts in combination with 16 reproducible histological features. The results of this discriminant analysis were compared with the results of the discriminant analyses in which only the histological features were used. RESULTS: Applying stepwise discriminant analysis, two histological features (an excess of histiocytes in the lamina propria and the villous or irregular aspect of the mucosal surface) in combination with IgMax were selected as the most discriminatory parameters that distinguish Crohn's disease from ulcerative colitis. IgMmax was defined as the maximum value of the mean percentage of IgM containing cells over all the biopsy locations. The use of this combination resulted in a better classification in 20% of the patients with Crohn's disease and in 9% of the patients with ulcerative colitis compared with the use of histological features alone. CONCLUSIONS: Morphometric enumeration of Ig subclass containing cells in colonic mucosal biopsy specimens has diagnostic value as a means of differentiating individual patients with Crohn's disease from those with ulcerative colitis. PMID:1556234
Guslandi, M; Sorghi, M; Tittobello, A
Twelve patients with dyspepsia whose gastric abnormalities ranged from diffuse reddening of the mucosa to multiple erosions were treated for 4 weeks with oral sulglycotide, a sulphated glycopeptide with known gastroprotective and ulcer-healing properties. Before and after treatment, gastric mucosal blood flow was assessed by means of laser Doppler flowmetry. A significant (P < 0.01) increase in mucosal perfusion was observed after sulglycotide treatment, suggesting that enhancement of mucosal blood flow may contribute to the therapeutic properties of the drug.
Wilson, Heather L; Obradovic, Milan R
The majority of lymphocytes activated at mucosal sites receive instructions to home back to the local mucosa, but a portion also seed distal mucosa sites. By seeding distal sites with antigen-specific effector or memory lymphocytes, the foundation is laid for the animal's mucosal immune system to respond with a secondary response should to this antigen be encountered at this site in the future. The common mucosal immune system has been studied quite extensively in rodent models but less so in large animal models such as the pig. Reasons for this paucity of reported induction of the common mucosal immune system in this species may be that distal mucosal sites were examined but no induction was observed and therefore it was not reported. However, we suspect that the majority of investigators simply did not sample distal mucosal sites and therefore there is little evidence of immune response induction in the literature. It is our hope that more pig immunologists and infectious disease experts who perform mucosal immunizations or inoculations on pigs will sample distal mucosal sites and report their findings, whether results are positive or negative. In this review, we highlight papers that show that immunization/inoculation using one route triggers mucosal immune system induction locally, systemically, and within at least one distal mucosal site. Only by understanding whether immunizations at one site triggers immunity throughout the common mucosal immune system can we rationally develop vaccines for the pig, and through these works we can gather evidence about the mucosal immune system that may be extrapolated to other livestock species or humans.
Thompson, Joseph M.; Whitmore, Alan C.; Konopka, Jennifer L.; Collier, Martha L.; Richmond, Erin M. B.; Davis, Nancy L.; Staats, Herman F.; Johnston, Robert E.
Vaccination represents the most effective control measure in the fight against infectious diseases. Local mucosal immune responses are critical for protection from, and resolution of, infection by numerous mucosal pathogens. Antigen processing across mucosal surfaces is the natural route by which mucosal immunity is generated, as peripheral antigen delivery typically fails to induce mucosal immune responses. However, we demonstrate in this article that mucosal immune responses are evident at multiple mucosal surfaces after parenteral delivery of Venezuelan equine encephalitis virus replicon particles (VRP). Moreover, coinoculation of null VRP (not expressing any transgene) with inactivated influenza virions, or ovalbumin, resulted in a significant increase in antigen-specific systemic IgG and fecal IgA antibodies, compared with antigen alone. Pretreatment of VRP with UV light largely abrogated this adjuvant effect. These results demonstrate that alphavirus replicon particles possess intrinsic systemic and mucosal adjuvant activity and suggest that VRP RNA replication is the trigger for this activity. We feel that these observations and the continued experimentation they stimulate will ultimately define the specific components of an alternative pathway for the induction of mucosal immunity, and if the activity is evident in humans, will enable new possibilities for safe and inexpensive subunit and inactivated vaccines. vaccine vector | Venezuelan equine encephalitis virus | viral immunology | RNA virus
Vitiñi, E; Alvarez, S; Medina, M; Medici, M; de Budeguer, M V; Perdigón, G
The beneficial properties of lactic acid bacteria (LAB) on human health have been frequently demonstrated. The interaction of LAB with the lymphoid cells associated to the gut to activate the mucosal immune system and the mechanisms by which they can exert an adjuvant effect is still unclear, as well as if this property is common for all the LAB. We studied the influence of the oral administration of different geneous of LAB such as Lactobacillus casei, L. acidophilus, L. rhamnosus, L. delbrueckii subsp. bulgaricus, L. plantarum, Lactococcus lactis and Streptococcus thermophilus. We determined if the LAB assayed were able to stimulate the specific, the non-specific immune response (inflammatory response), or both. We demonstrated that all the bacteria assayed were able to increase the number of IgA producing cells associated to the lamina propria of small intestine. This effect was dose dependent. The increase in IgA+ producing cells was not always correlated with an increase in the CD4+ T cell number, indicating that some LAB assayed only induced clonal expansion of B cells triggered to produce IgA. Most of them, induced an increase in the number of cells involved in the inflammatory immune response. CD8+ T cell were diminished or not affected, with exception of L. plantarum that induced an increase at low dose. This fact would mean that LAB are unable to induce cytotoxicity mechanisms. We demonstrated the importance in the selection of LAB to be used as gut mucosal adjuvant. The different behaviours observed among them on the gut mucosal immune response, specially those that induce inflammatory immune response, show that not all the LAB can be used as oral adjuvant and that the beneficial effect of them can not generalized to genous or specie. The immunoadjuvant capacity would be a property of the strain assayed.
Li, Qingsheng; Estes, Jacob D.; Schlievert, Patrick M.; Duan, Lijie; Brosnahan, Amanda J.; Southern, Peter J.; Reilly, Cavan S.; Peterson, Marnie L.; Schultz-Darken, Nancy; Brunner, Kevin G.; Nephew, Karla R.; Pambuccian, Stefan; Lifson, Jeffrey D.; Carlis, John V.; Haase, Ashley T.
While there has been great progress in treating HIV-1 infection1, preventing transmission has thus far proven an elusive goal. Indeed, recent trials of a candidate vaccine and microbicide have been disappointing, both for want of efficacy and concerns about increased rates of transmission2–4. Nonetheless, studies of vaginal transmission in the SIV-rhesus macaque model point to opportunities in the earliest stages of infection where a vaccine or microbicide might be protective, by limiting the expansion of infected founder populations at the portal of entry5, 6. Here we show in this SIV-macaque model, that an outside-in endocervical mucosal signalling system, involving MIP-3α, plasmacytoid dendritic cells and CCR5+cell-attracting chemokines produced by these cells, in combination with the innate immune and inflammatory responses to infection in both cervix and vagina, recruit CD4+T cells to fuel this obligate expansion. We then show that glycerol monolaurate, a widely used antimicrobial compound 7 with inhibitory activity against production of MIP-3α and other proinflammatory cytokines8, can inhibit mucosal signalling and the innate and inflammatory response to HIV-1 and SIV in vitro, and in vivo can protect rhesus macaques from acute infection despite repeated intra-vaginal exposure to high doses of SIV. This novel approach, plausibly linked to interfering with innate host responses that recruit the target cells necessary to establish systemic infection, opens a promising new avenue for development of effective interventions to block HIV-1 mucosal transmission. PMID:19262509
Computational modeling techniques are playing increasingly important roles in advancing a systems-level mechanistic understanding of biological processes. Computer simulations guide and underpin experimental and clinical efforts. This study presents ENteric Immune Simulator (ENISI), a multiscale modeling tool for modeling the mucosal immune responses. ENISI's modeling environment can simulate in silico experiments from molecular signaling pathways to tissue level events such as tissue lesion formation. ENISI's architecture integrates multiple modeling technologies including ABM (agent-based modeling), ODE (ordinary differential equations), SDE (stochastic modeling equations), and PDE (partial differential equations). This paper focuses on the implementation and developmental challenges of ENISI. A multiscale model of mucosal immune responses during colonic inflammation, including CD4+ T cell differentiation and tissue level cell-cell interactions was developed to illustrate the capabilities, power and scope of ENISI MSM. Background Computational techniques are becoming increasingly powerful and modeling tools for biological systems are of greater needs. Biological systems are inherently multiscale, from molecules to tissues and from nano-seconds to a lifespan of several years or decades. ENISI MSM integrates multiple modeling technologies to understand immunological processes from signaling pathways within cells to lesion formation at the tissue level. This paper examines and summarizes the technical details of ENISI, from its initial version to its latest cutting-edge implementation. Implementation Object-oriented programming approach is adopted to develop a suite of tools based on ENISI. Multiple modeling technologies are integrated to visualize tissues, cells as well as proteins; furthermore, performance matching between the scales is addressed. Conclusion We used ENISI MSM for developing predictive multiscale models of the mucosal immune system during gut
Li, Qingsheng; Estes, Jacob D; Schlievert, Patrick M; Duan, Lijie; Brosnahan, Amanda J; Southern, Peter J; Reilly, Cavan S; Peterson, Marnie L; Schultz-Darken, Nancy; Brunner, Kevin G; Nephew, Karla R; Pambuccian, Stefan; Lifson, Jeffrey D; Carlis, John V; Haase, Ashley T
Although there has been great progress in treating human immunodeficiency virus 1 (HIV-1) infection, preventing transmission has thus far proven an elusive goal. Indeed, recent trials of a candidate vaccine and microbicide have been disappointing, both for want of efficacy and concerns about increased rates of transmission. Nonetheless, studies of vaginal transmission in the simian immunodeficiency virus (SIV)-rhesus macaque (Macacca mulatta) model point to opportunities at the earliest stages of infection in which a vaccine or microbicide might be protective, by limiting the expansion of infected founder populations at the portal of entry. Here we show in this SIV-macaque model, that an outside-in endocervical mucosal signalling system, involving MIP-3alpha (also known as CCL20), plasmacytoid dendritic cells and CCR5(+ )cell-attracting chemokines produced by these cells, in combination with the innate immune and inflammatory responses to infection in both cervix and vagina, recruits CD4(+) T cells to fuel this obligate expansion. We then show that glycerol monolaurate-a widely used antimicrobial compound with inhibitory activity against the production of MIP-3alpha and other proinflammatory cytokines-can inhibit mucosal signalling and the innate and inflammatory response to HIV-1 and SIV in vitro, and in vivo it can protect rhesus macaques from acute infection despite repeated intra-vaginal exposure to high doses of SIV. This new approach, plausibly linked to interfering with innate host responses that recruit the target cells necessary to establish systemic infection, opens a promising new avenue for the development of effective interventions to block HIV-1 mucosal transmission.
Purpose of review Significant advances have been made in the characterization of Vitamin D and the Vitamin D receptor (VDR) in immune function. The studies of signaling pathways involved in the response to infection and inflammation have led to a more detailed understanding of the cellular response to Vitamin D through VDR. This review summarizes recent progress in understanding how Vitamin D contributes to mucosal immune function, particularly in relation to the molecular mechanisms by which Vitamin D and VDR influence mucosal immunity, bacterial infection, and inflammation. Recent findings Recently, it was shown that Vitamin D modulates the T cell antigen receptor, further demonstrating that Vitamin D has a nonclassical role in immunoregulation. The anti-inflammation and anti-infection functions for Vitamin D are newly identified and highly significant activities. Vitamin D/VDR have multiple critical functions in regulating the response to intestinal homeostasis, tight junctions, pathogen invasion, commensal bacterial colonization, antimicrobe peptide secretion, and mucosal defense. Interestingly, microorganisms modulate the VDR signaling pathway. Summary Vitamin D is known as a key player in calcium homeostasis and electrolyte and blood pressure regulation. Recently, important progress has been made in understanding how the noncanonical activities of Vitamin D influence the pathogenesis and prevention of human disease. Vitamin D and VDR are directly involved in T cell antigen receptor signaling. The involvement of Vitamin D/VDR in anti-inflammation and anti-infection represents a newly identified and highly significant activity for VDR. Studies have indicated that the dysregulation of VDR may lead to exaggerated inflammatory responses, raising the possibility that defects in Vitamin D and VDR signaling transduction may be linked to bacterial infection and chronic inflammation. Further characterization of Vitamin D/VDR will help elucidate the pathogenesis of
Raufman, J. P.
This contribution reviews briefly the history of the discovery and characterization of peptic activity; secretory models and current concepts regarding the regulation of pepsinogen secretion; and evidence that pepsin is a necessary co-factor for gastroduodenal mucosal injury. Several animal studies indicate that peptic activity is required for acid- and nonsteroidal anti-inflammatory drug-induced gastroduodenal ulceration. A more vigorous approach to the development of anti-peptic drugs for the treatment of peptic ulcer disease is encouraged. Images Figure 1 PMID:9041694
Wang, Wen-Hung; Lin, Yen-Chun; Chen, Wen-Cheng; Chen, Miao-Fen; Chen, Chih-Cheng; Lee, Kam-Fai
Purpose: This study evaluated the feasibility of screening mucosal recurrent nasopharyngeal carcinoma with narrow-band imaging (NBI) endoscopy. Methods and Materials: One hundred and six patients were enrolled. All patients underwent conventional white-light (WL) endoscopic examination of the nasopharynx followed by NBI endoscopy. Biopsies were performed if recurrence was suspected. Results: We identified 32 suspected lesions by endoscopy in WL and/or NBI mode. Scattered brown spots (BS) were identified in 22 patients, and 4 of the 22 who had negative MRI findings were histopathologically confirmed to be neoplasias that were successfully removed via endoscopy. A comparison of the visualization in NBI closer view corresponded to histopathological findings in 22 BS, and the prevalence rates of neoplasias in tail signs, round signs, and irregularities signs were 0% (0/6), 0% (0/7), and 44.4% (4/9), respectively (p = 0.048). The sensitivity, specificity, and diagnostic capability were 37.5%, 92.9% and 0.652 for WL, 87.5%, 74.5% and 0.810 for NBI, and 87.5%, 87.8%, and 0.876 for NBI closer view, respectively. NBI closer view was effective in increasing specificity compared with NBI alone (87.8% vs. 74.5%, p < 0.05), and in increasing sensitivity and diagnostic capability compared to WL alone (87.5% vs. 37.5%, p < 0.05; 0.876 vs. 0.652, p = 0.0001). Conclusions: Although NBI in endoscopy can improve sensitivity of mucosal recurrent nasopharyngeal neoplasias, false-positive (nonneoplasia BS) results may be obtained in areas with nonspecific inflammatory changes due to postradiation effects. NBI closer view not only can offer a timely, convenient, and highly reliable assessment of mucosal recurrent nasopharyngeal carcinoma, it can also make endoscopic removal possible.
Van Damme, N; De Vos, M; Baeten, D; Demetter, P; Mielants, H; Verbruggen, G; Cuvelier, C; Veys, E; De Keyser, F
OBJECTIVE—To quantify the fraction of gut mucosal lymphocytes expressing the T helper type 1 (Th1) cytokines, interferon γ (IFNγ) and interleukin (IL)2, and the Th2 cytokines, IL4 and IL10, at the single cell level in patients with spondyloarthropathy (SpA) in comparison with healthy controls. METHODS—An improved extraction protocol was used for the enrichment of intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) from colonic and ileal biopsy specimens obtained from patients with SpA (n=20) and healthy controls (n=13). After stimulation with phorbol ester/ionomycin, expression of the intracellular cytokines IFNγ, IL2, IL4, and IL10 was determined in CD3+, CD3+CD8+ and CD3+CD8− T cells by flow cytometry. RESULTS—In colonic LPLs, a significant decrease in IFNγ-producing CD3+ cells was observed (p=0.02) in patients with SpA. In the CD3+CD8− subset, the proportion of cells producing IFNγ and IL2 was decreased in patients with SpA (p=0.021 and p=0.027 respectively). In ileal LPLs, the percentage of IL10-producing CD3+CD8− cells was significantly increased (p=0.046). CONCLUSION—An impaired Th1 cytokine profile is observed in gut mucosal lymphocytes from patients with SpA. This adds to the existing evidence that the gut mucosal immune apparatus is involved in the pathogenesis of SpA. PMID:11302872
Oxholm, P; Manthorpe, R; Oxholm, A; Schiødt, M
Lower lip biopsies from twenty-three consecutive patients under evaluation for Sjögren's syndrome, and from six normal controls, were investigated for deposits of immunoglobulins, fibrinogen and C3, using a direct immunofluorescence technique. Deposits of both IgG and IgA were demonstrated in the mucosal epithelium in three of six patients with primary Sjögren's syndrome. Similar IgG deposits were found in two of three patients with xerostomia and in one of three patients with Sjögren's syndrome secondary to rheumatoid arthritis. Immunoglobulins were located in close relation to cell surfaces in the basal and suprabasal layers of the epithelium. Double labelling experiments indicated a partial topographic concordance between the immunoglobulin deposits and OKT6 positive Langerhans cells in the epithelium. No deposits of immunoglobulins, fibrinogen or C3 were found in the remaining eleven patients and six normal controls. We conclude that deposits of IgG and IgA in the labial mucosal epithelium seem to be a characteristic finding in patients with primary Sjögren's syndrome as well as in patients with xerostomia. The diagnostic value of this new observation needs to be clarified in future studies.
Yokokura, Hideto; Demitsu, Toshio; Kakurai, Maki; Umemoto, Naoka; Azuma, Ryuichi; Yamada, Tomoko; Suzuki, Masayuki; Jimbu, Yoshinori; Yoneda, Kozo; Ishii, Norito; Hashimoto, Takashi
A 58-year-old Japanese male visited us with painful lesions on the lower lip, oral mucosa and genital region of an 8-month duration. Histological features of the genital lesion were almost consistent with lichenoid tissue reaction. A few intraepidermal acantholytic keratinocytes were also seen in the suprabasal clefts. Direct immunofluorescence exhibited cell surface immunoglobulin (Ig)G deposition and linear deposition of fibrinogen at the dermoepidermal junction. IgG anti-desmoglein (Dsg)3 antibody, but not anti-Dsg1 antibody, was detected in the patient's serum by enzyme-linked immunosorbent assay. Immunoblotting using normal human epidermal extract detected the 210-kD envoplakin, 190-kD periplakin and 130-kD Dsg3. The diagnosis of paraneoplastic pemphigus (PNP) was made. Subsequent investigation revealed a large space-occupying lesion in the liver. Histological findings from liver biopsy specimen were consistent with hepatocellular carcinoma. The patient has been alive 38 months after the diagnosis of PNP was made, although the liver mass has slowly enlarged. Our case is clinically and histologically similar to erosive mucosal lichen planus. Immunological studies confirmed the diagnosis of PNP. The results of negative Dsg1 and positive Dsg3 were consistent with clinical features showing severe mucosal involvement without cutaneous erosion. In PNP, the association with non-hematological solid tumor is extremely rare.
Jayatilake, J A M S; Samaranayake, Y H; Samaranayake, L P
The purpose of this study is to compare the light and scanning electron microscopic (SEM) features of tissue invasion by three Candida species (C. albicans, C. tropicalis, and C. dubliniensis) in two different tissue culture models: rabbit tongue mucosal explants (RTME) and reconstituted human oral epithelium (RHOE). Tongue mucosal biopsies of healthy New Zealand rabbits were maintained in explant culture using a transwell system. RHOE was obtained from Skinethic Laboratory (Nice, France). RTME and RHOE were inoculated with C. albicans, C. tropicalis, and C. dubliniensis separately and incubated at 37 degrees C, 5% CO(2), and 100% humidity up to 48 h. Light microscopic and SEM examinations of uninfected (controls) and infected tissues were performed at 24 and 48 h. C. albicans produced characteristic hallmarks of pathological tissue invasion in both tissue models over a period of 48 h. Hyphae penetrated through epithelial cells and intercellular gaps latter resembling thigmotropism. SEM showed cavitations on the epithelial cell surfaces particularly pronounced at sites of hyphal invasion. Some hyphae on RTME showed several clusters of blastospores attached in regular arrangements resembling "appareil sporifere". C. tropicalis and C. dubliniensis produced few hyphae mainly on RTME but they did not penetrate either model. Our findings indicate that multiple host-fungal interactions such as cavitations, thigmotropism, and morphogenesis take place during candidal tissue invasion. RTME described here appears to be useful in investigations of such pathogenic processes of Candida active at the epithelial front.
Bajic, Juliana E; Eden, Georgina L; Lampton, Lorrinne S; Cheah, Ker Y; Lymn, Kerry A; Pei, Jinxin V; Yool, Andrea J; Howarth, Gordon S
AIM To investigate the effects of orally gavaged aqueous rhubarb extract (RE) on 5-fluorouracil (5-FU)-induced intestinal mucositis in rats. METHODS Female Dark Agouti rats (n = 8/group) were gavaged daily (1 mL) with water, high-dose RE (HDR; 200 mg/kg) or low-dose RE (LDR; 20mg/kg) for eight days. Intestinal mucositis was induced (day 5) with 5-FU (150 mg/kg) via intraperitoneal injection. Intestinal tissue samples were collected for myeloperoxidase (MPO) activity and histological examination. Xenopus oocytes expressing aquaporin 4 water channels were prepared to examine the effect of aqueous RE on cell volume, indicating a potential mechanism responsible for modulating net fluid absorption and secretion in the gastrointestinal tract. Statistical significance was assumed at P < 0.05 by one-way ANOVA. RESULTS Bodyweight was significantly reduced in rats administered 5-FU compared to healthy controls (P < 0.01). Rats administered 5-FU significantly increased intestinal MPO levels (≥ 307%; P < 0.001), compared to healthy controls. However, LDR attenuated this effect in 5-FU treated rats, significantly decreasing ileal MPO activity (by 45%; P < 0.05), as compared to 5-FU controls. 5-FU significantly reduced intestinal mucosal thickness (by ≥ 29% P < 0.001) as compared to healthy controls. LDR significantly increased ileal mucosal thickness in 5-FU treated rats (19%; P < 0.05) relative to 5-FU controls. In xenopus oocytes expressing AQP4 water channels, RE selectively blocked water influx into the cell, induced by a decrease in external osmotic pressure. As water efflux was unaltered by the presence of extracellular RE, the directional flow of water across the epithelial barrier, in the presence of extracellular RE, indicated that RE may alleviate water loss across the epithelial barrier and promote intestinal health in chemotherapy-induced intestinal mucositis. CONCLUSION In summary, low dose RE improves selected parameters of mucosal integrity and reduces ileal
Vaccination is the most successful immunological practice that improves the quality of human life and health. Vaccine materials include antigens of pathogens and adjuvants potentiating the effectiveness of vaccination. Vaccines are categorized using various criteria, including the vaccination material used and the method of administration. Traditionally, vaccines have been injected via needles. However, given that most pathogens first infect mucosal surfaces, there is increasing interest in the establishment of protective mucosal immunity, achieved by vaccination via mucosal routes. This review summarizes recent developments in mucosal vaccines and their associated adjuvants. PMID:28168169
Cirakoglu, Abdullah; Ogreden, Ercan; Cetinkol, Yeliz; Calgin, Mustafa Kerem; Ayyildiz, Ali
Aim. The aim of this study is to evaluate whether leaving the biopsy needle used during prostate needle biopsy in 10% povidone-iodine (betadine) solution affects the infectious complications forming after biopsy. Material and Method. This study retrospectively evaluated the data of 176 patients with prostate biopsy performed between December 2012 and April 2014. Patients in Group 1 (n = 89) were given ofloxacin as a prophylactic antibiotic before biopsy. Patients in Group 2 (n = 87) had the biopsy needle left in povidone-iodine solution for 1 minute before each use, in addition to antibiotic prophylaxis. The two groups were compared in terms of infective complications developing after biopsy. Results were analyzed using the Mann–Whitney U test and Fisher's exact test. Results. The distribution of infective complications after biopsy according to group was as follows. Group 1, not using betadine, had 15.7% fever, 13.5% hospital stay, 12.4% urinary retention, 10.1% prostatitis, and 5.6% sepsis. The distribution of the same complications in Group 2 using betadine was identified as 5.7% fever, 4.6% hospital stay, 3.4% urinary retention, 2.3% prostatitis, and 0% sepsis. The use of betadine was found to significantly reduce the infectious complications after biopsy compared to the control group (p < 0.05). Conclusion. At the end of this study leaving the prostate needle in povidone-iodine solution before each use during prostate biopsy was found to reduce the infective complications and hospital stay after biopsy. PMID:28096812
Shih, W.J.; DeLand, F.H.; Domstad, P.A.; Magoun, S.; Dillon, M.L.
When abnormally increased radioactivity is seen in a rib or ribs by bone imaging in a patient with suspected or known malignancy, it frequently is difficult to differentiate fracture from metastatic disease. Histological examination of the lesion is crucial for diagnosis, staging, and planning of therapy. To assess the value of external localization of the site or sites of abnormal uptake in a rib as a guide for open rib biopsy, 10 patients (7 men, 3 women; age range, 34 to 68 years) with known or suspected malignancy were studied. With reference to the oscilloscope image, a cobalt 57 marker was placed on the skin overlying the focus of increased uptake, and the area of increased activity was marked on the skin as a guide to surgical resection. Of ten resected ribs, four showed metastatic disease and five had fractures. (One patient underwent two external marking procedures and two surgical procedures.) Rib biopsy was not performed in 1 patient because prior to the surgical procedure, a small subcutaneous nodule adjacent to the skin marker was excised and confirmed to be carcinoma. Appropriate courses of management (operation, irradiation, chemotherapy) were taken after the biopsies. The surgeon responsible for the biopsy should be present during the skin-marking procedure, and the area beneath the scapula and the region adjacent to the spine should be avoided. Our results indicate that the technique is a very useful aid for approaching open rib biopsies more precisely.
Alexander, J A; Smith, B J
Control of patient respiration is needed to safely perform percutaneous liver biopsy (PLB) and may be adversely affected by sedation. The purpose of this study was to evaluate the safety of PLB with intravenous midazolam and to evaluate patient acceptance of PLB with and without sedation. Two hundred seventeen consecutive patients underwent 301 percutaneous liver biopsies. One hundred fifty-one of the biopsies were done after the patients were sedated with intravenous midazolam immediately before the biopsy. The last 61 patients were questioned after the biopsy to evaluate the discomfort of the procedure, their memory of the procedure, and their willingness to undergo another PLB. The major complication rate was similar in the midazolam-treated (0.7%) and untreated (0.7%) groups. The midazolam-treated patients had a numerically lower mean pain score (1.5 +/- 0.4 vs 4.0 +/- 0.7) (mean +/- SEM) (P = 0.07) and significantly lower mean memory score (4.8 +/- 0.7 vs 9.9 +/- 0.1) (P < 0.01) than the untreated patients. The treated and untreated groups had similar mean willingness for repeat PLB scores (9.3 +/- 0.3 vs 9.1 +/- 0.6). We conclude that: (1) there is no increased risk of PLB with midazolam and (2) patients have less memory of the procedure with midazolam.
Steel, S. J.; Winstanley, D. P.
During the last five years 140 biopsies of the lung or pleura have been performed with a high-speed air-drill. A specially designed trephine, 2·1 mm. in bore, proved effective in obtaining adequate specimens from patients with diffuse lung lesions. With this method 111 out of 119 (93%) consecutive lung biopsies were successful in producing specimens of lung tissue for histological examination and 101 (85%) of these biopsies were diagnostically significant. Conditions such as sarcoidosis, berylliosis, alveolar proteinosis, polyarteritis nodosa, asbestosis, and diffuse interstitial pulmonary fibrosis were conclusively diagnosed. Complications were not serious and the commonest, pneumothorax, which occurred in 26% of the lung biopsies, was usually asymptomatic. The procedure is rapid and painless and breath-holding is unnecessary. It is performed under local anaesthesia and can be repeated, if required, thus having many advantages over biopsy by open thoracotomy. However, full thoracic surgical cover should be available. Indications include diffuse and localized lesions of the lung or pleura where a definitive diagnosis is required for the purpose of treatment, prognosis, or a claim for industrial compensation. Images PMID:5348325
Laredo, Jean-Denis Hamze, Bassam; Jeribi, Riadh
Biopsy is usually performed as the first step in percutaneous treatment of osteoid osteomas prior to laser photocoagulation. At our institution, 117 patients with a presumed diagnosis of osteoid osteoma had a trephine biopsy before a percutaneous laser photocoagulation. Biopsies were made using two different types of needles. A Bonopty biopsy needle (14-gauge cannula, 16-gauge trephine needle; Radi Medical Systems, Uppsala, Sweden) was used in 65 patients, and a Laurane biopsy needle (11-gauge cannula, 12.5-gauge trephine needle; Laurane Medical, Saint-Arnoult, France) in 43 patients. Overall biopsy results were positive for osteoid osteoma in 83 (70.9%) of the 117 cases. The Laurane needle provided a significantly higher positive rate (81.4%) than the Bonopty needle (66.1%; p < 0.05). This difference was not due to the size of the nidus, which was similar in the two groups (p < 0.05) and may be an effect of differences in needle caliber (12.5 vs. 14 gauge) as well as differences in needle design. The rate of positive biopsy results obtained in the present series with the Laurane biopsy needle is, to our knowledge, the highest rate reported in series dealing with percutaneous radiofrequency ablation and laser photocoagulation of osteoid osteomas.
Kawaai, Hiroyoshi; Yoshida, Kenji; Tanaka, Eri; Togami, Kohei; Tada, Hitoshi; Ganzberg, Steven; Yamazaki, Shinya
There is an abundance of blood vessels in the oral cavity, and intraoperative bleeding can disrupt operations. There have been some interesting reports about constriction of vessels in the oral cavity, one of which reported that gingival blood flow in cats is controlled by sympathetic α-adrenergic fibres that are involved with vasoconstriction. Dexmedetomidine is a sedative and analgesic agent that acts through the α-2 adrenoceptor, and is expected to have a vasoconstrictive action in the oral cavity. We have focused on the relation between the effects of α-adrenoceptors by dexmedetomidine and vasoconstriction in oral tissues, and assessed the oral mucosal blood flow during sedation with dexmedetomidine. The subjects comprised 13 healthy male volunteers, sedated with dexmedetomidine in a loading dose of 6 μg/kg/h for 10 min and a continuous infusion of 0.7 μg/kg/h for 32 min. The mean arterial pressure (MAP), heart rate (HR), cardiac output (CO), stroke volume (SV), systemic vascular resistance (SVR), and palatal mucosal blood flow (PMBF) were measured at 0, 5, 10, 12, 22, and 32 min after the start of the infusion. The HR, CO, and PBMF decreased significantly during the infusion even though there were no differences in the SV. The SVR increased significantly but the PMBF decreased significantly. In conclusion, PMBF was reduced by the mediating effect of dexmedetomidine on α-2 adrenoceptors.
Aujla, Shean J.; Dubin, Patricia J.; Kolls, Jay K.
Th17 cells are a new lineage of T-cells that are controlled by the transcription factor RORγt and develop independent of GATA-3, T-bet, Stat 4 and Stat 6. Novel effector molecules produced by these cells include IL-17A, IL-17F, IL-22, and IL-26. IL-17RA binds IL-17A and IL-17F and is critical for host defense against extracellular planktonic bacteria by regulating chemokine gradients for neutrophil emigration into infected tissue sites as well as host granulopoiesis. Moreover IL-17 and IL-22 regulate the production of antimicrobial proteins in mucosal epithelium. Although TGF-β1 and IL-6 have been shown to be critical for development of Th17 cells from naïve precursors, IL-23 is also important in regulating IL-17 release in mucosal tissues in response to infectious stimuli. Compared to Th1 cells, IL-23 and IL-17 show limited roles in controlling host defense against primary infections with intracellular bacteria such as Mycobacterium tuberculosis suggesting a predominate role of the Th17 lineage in host defense against extracellular pathogens. However in the setting of chronic biofilm infections, as that occurs with Cystic Fibrosis or bronchetctasis, Th17 cells may be key contributors of tissue injury. PMID:18054248
Harris, R Alan; Nagy-Szakal, Dorottya; Mir, Sabina A V; Frank, Eibe; Szigeti, Reka; Kaplan, Jess L; Bronsky, Jiri; Opekun, Antone; Ferry, George D; Winter, Harland; Kellermayer, Richard
Inflammatory bowel diseases (IBD) are emerging globally, indicating that environmental factors may be important in their pathogenesis. Colonic mucosal epigenetic changes, such as DNA methylation, can occur in response to the environment and have been implicated in IBD pathology. However, mucosal DNA methylation has not been examined in treatment-naïve patients. We studied DNA methylation in untreated, left sided colonic biopsy specimens using the Infinium HumanMethylation450 BeadChip array. We analyzed 22 control (C) patients, 15 untreated Crohn's disease (CD) patients, and 9 untreated ulcerative colitis (UC) patients from two cohorts. Samples obtained at the time of clinical remission from two of the treatment-naïve UC patients were also included into the analysis. UC-specific gene expression was interrogated in a subset of adjacent samples (5 C and 5 UC) using the Affymetrix GeneChip PrimeView Human Gene Expression Arrays. Only treatment-naïve UC separated from control. One-hundred-and-twenty genes with significant expression change in UC (> 2-fold, P<0.05) were associated with differentially methylated regions (DMRs). Epigenetically associated gene expression changes (including gene expression changes in the IFITM1, ITGB2, S100A9, SLPI, SAA1, and STAT3 genes) were linked to colonic mucosal immune and defense responses. These findings underscore the relationship between epigenetic changes and inflammation in pediatric treatment-naïve UC and may have potential etiologic, diagnostic, and therapeutic relevance for IBD.
Dalakas, Marinos C
The inflammatory myopathies encompass a heterogeneous group of acquired muscle diseases characterized clinically, by muscle weakness, and histologically, by inflammatory infiltrates within the skeletal muscles. The group of these myopathies comprise three major and discrete subsets: polymyositis (PM), dermatomyositis (DM), and inclusion body myositis (IBM). Each subset retains its characteristic clinical, immunopathologic, and morphologic features regardless of whether it occurs separately or in connection with other systemic diseases. Although the diagnosis of these disorders is based on the combination of clinical examination, electromyographic data, serum muscle enzyme levels, various autoantibodies, and the muscle biopsy findings, the muscle biopsy offers the most definitive diagnostic information in the majority of the cases. This article summarizes the main histologic features that characterize PM, DM, or IBM and emphasizes the main pitfalls associated with interpretation of the biopsies.
Whittier, William L
Percutaneous kidney biopsy is an integral part of a nephrologist's practice. It has helped to define nephrology as a subspecialty. When indicated, it is a necessary procedure to help patients, as it allows for diagnostic, prognostic, and therapeutic information. Although very safe, this procedure can give rise to complications, mainly related to bleeding. Since its development in the 1950s, modifications have been made to the approach and the technique, which have improved the diagnostic yield while keeping it a safe procedure. Alterations to the standard approach may be necessary if risk factors for bleeding are present. In addition, obesity, pregnancy, and solitary kidney biopsy are all special circumstances that change the procedure itself or the risk of the procedure. Today, kidney biopsy is a vital procedure for the nephrologist: clinically relevant, safe, and effective.
Faries, Mark B; Morton, Donald L
In patients with melanoma, surgery is pivotal not only for the primary tumor but also for regional and often distant metastases. The minimally invasive technique of sentinel node (SN) biopsy has become standard for detection of occult regional node metastasis in patients with intermediate-thickness primary melanoma; in these patients it has a central role in determining prognosis and a significant impact on survival when biopsy results are positive. Its role in thin melanoma remains under evaluation. The regional tumor-draining SN also is a useful model for studies of melanoma-induced immunosuppression. Although completion lymphadenectomy remains the standard of care for patients with SN metastasis, results of ongoing phase III trials will indicate whether SN biopsy without further lymph node surgery is adequate therapy for certain patients with minimal regional node disease.
Jia, T; Xing, L; Zhu, F; Jin, X; Liu, L; Tao, J; Chen, Y; Gao, Z; Zhang, H
We have developed a new method for minimally-invasive treatment of uncomplicated oral ranulas using a mucosal tunnel, and we report the clinical outcome. We constructed a mucosal tunnel for each of 35 patients who presented with an oral ranula, by making 2 parallel incisions across the top of the protruding ranula 2-3mm apart, and dissected the soft tissue along the incisions to its wall. The fluid was removed and the cavity irrigated with normal saline. The wall of the ranula was not treated. The first mucosal tunnel was made by suturing the base of the mucosal strip to the deepest part of the wall of the ranula. The mucosal base of the tunnel and the deepest part of the base of the ranula were fixed with absorbable sutures. The two external edges of the incisions were sutured together to form the second mucosal tunnel, and apposing sutures were inserted between the two parallel incisions to form two natural mucosal tunnels. The duration of follow-up ranged from 1 to 5 years. One patient was lost to follow-up and 34 patients were cured. Outcomes were satisfactory without relapse during the follow-up period and the patients were satisfied with the outcome. The mucosal tunnel is a safe, effective, simple, and minimally-invasive treatment for oral ranula.
Su, Fei; Patel, Girishchandra B.; Hu, Songhua; Chen, Wangxue
ABSTRACT Generation of protective immunity at mucosal surfaces can greatly assist the host defense against pathogens which either cause disease at the mucosal epithelial barriers or enter the host through these surfaces. Although mucosal routes of immunization, such as intranasal and oral, are being intensely explored and appear promising for eliciting protective mucosal immunity in mammals, their application in clinical practice has been limited due to technical and safety related challenges. Most of the currently approved human vaccines are administered via systemic (such as intramuscular and subcutaneous) routes. Whereas these routes are acknowledged as being capable to elicit antigen-specific systemic humoral and cell-mediated immune responses, they are generally perceived as incapable of generating IgA responses or protective mucosal immunity. Nevertheless, currently licensed systemic vaccines do provide effective protection against mucosal pathogens such as influenza viruses and Streptococcus pneumoniae. However, whether systemic immunization induces protective mucosal immunity remains a controversial topic. Here we reviewed the current literature and discussed the potential of systemic routes of immunization for the induction of mucosal immunity. PMID:26752023
Gladkova, Natalia; Maslennikova, Anna; Terentieva, Anna; Fomina, Yulia; Khomutinnikova, Nina; Balalaeva, Irina; Vyseltseva, Yulia; Larin, Roman; Kornoukhova, Natalia; Shakhov, Andrey; Shakhova, Natalia; Gelikonov, Grigory; Kamensky, Vladislav; Feldchtein, Felix
We present pilot results in optical coherence tomography (OCT) visualization of normal mucosa radiation damage. 15 patients undergoing radiation treatment of head and neck cancer were enrolled. OCT was used to monitor the mucositis development during and after treatment. OCT can see stages of radiation mucositis development, including hidden ones, before any clinical manifestations.
Marsh, Michael N.; Heal, Calvin J.
The evolving history of the small intestinal biopsy and its interpretation—and misinterpretations—are described in this paper. Certain interpretative errors in the technical approaches to histological assessment are highlighted—even though we may never be rid of them. For example, mucosal “flattening” does not reduce individual villi to their cores, as still seems to be widely believed. Neither is the mucosa undergoing an atrophic process—since it can recover structurally. Rather, the intestinal mucosa manifests a vast hypertrophic response resulting in the formation of large plateaus formed from partially reduced villi and their amalgamation with the now increased height and width of the inter-villous ridges: this is associated with considerable increases in crypt volumes. Sections through mosaic plateaus gives an erroneous impression of the presence of stunted, flat-topped villi which continues to encourage both the continued use of irrelevant “atrophy” terminologies and a marked failure to perceive what random sections through mosaic plateaus actually look like. While reviewing the extensive 40+ year literature on mucosal analysis, we extracted data on intraepithelial lymphocytes (IEL) counts from 607 biopsies, and applied receiver-operating characteristic (ROC)-curve analysis. From that perspective, it appears that counting IEL/100 enterocyte nuclei in routine haematoxylin and eosin (H&E) sections provides the most useful discriminator of celiac mucosae at histological level, with an effective cut-off of 27 IEL, and offering a very high sensitivity with few false negatives. ROC-curve analysis also revealed the somewhat lesser accuracies of either CD3+ or γδ+ IEL counts. Current official guidelines seem to be somewhat inadequate in clearly defining the spectrum of gluten-induced mucosal pathologies and how they could be optimally interpreted, as well as in promoting the ideal manner for physicians and pathologists to interact in interpreting
Shimamoto, Hiroshi Inaba, Yoshitaka; Yamaura, Hidekazu; Sato, Yozo; Kamiya, Mika; Miyazaki, Masaya; Arai, Yasuaki; Horio, Yoshitsugu
We described a case of chest wall dissemination after percutaneous transthoracic needle biopsy. A 65-year-old man had a lung nodule which was suspected to be lung carcinoma. He underwent percutaneous transthoracic needle biopsy using an 18G semiautomated biopsy needle and pathologic diagnosis showed organizing pneumonia. Two months after the biopsy, chest wall dissemination occurred. Implantation of carcinoma along the biopsy route was suspected, but the mass was actually due to pulmonary nocardiosis.
Pavot, Vincent; Rochereau, Nicolas; Lawrence, Philip; Girard, Marc P; Genin, Christian; Verrier, Bernard; Paul, Stéphane
In spite of several attempts over many years at developing a HIV vaccine based on classical strategies, none has convincingly succeeded to date. As HIV is transmitted primarily by the mucosal route, particularly through sexual intercourse, understanding antiviral immunity at mucosal sites is of major importance. An ideal vaccine should elicit HIV-specific antibodies and mucosal CD8⁺ cytotoxic T-lymphocyte (CTL) as a first line of defense at a very early stage of HIV infection, before the virus can disseminate into the secondary lymphoid organs in mucosal and systemic tissues. A primary focus of HIV preventive vaccine research is therefore the induction of protective immune responses in these crucial early stages of HIV infection. Numerous approaches are being studied in the field, including building upon the recent RV144 clinical trial. In this article, we will review current strategies and briefly discuss the use of adjuvants in designing HIV vaccines that induce mucosal immune responses.
Burgener, Adam; McGowan, Ian; Klatt, Nichole R
The mucosal barrier plays an integral function in human health as it is the primary defense against pathogens, and provides a critical transition between the external environment and the human internal body. In the context of HIV infection, the most relevant mucosal surfaces include those of the gastrointestinal (GI) and genital tract compartments. Several components help maintain the effectiveness of this mucosal surface, including the physical anatomy of the barrier, cellular immunity, soluble factors, and interactions between the epithelial barrier and the local microenvironment, including mucus and host microbiota. Any defects in barrier integrity or function can rapidly lead to an increase in acquisition risk, or with established infection may result in increased pathogenesis, morbidities, or mortality. Indeed, a key feature to all aspects of HIV infection from transmission to pathogenesis is disruption and/or dysfunction of mucosal barriers. Herein, we will detail the host-pathogen relationship of HIV and mucosal barriers in both of these scenarios.
Gorriz, Elias; Reyes, Ricardo; Lobrano, Mary Beth; Pulido-Duque, Juan M.; San Roman, Jose L.; Lonjedo, Elena; Ferral, Hector; Maynar, Manuel
Seventy-seven transjugular liver biopsies were performed with a coaxial, spring-loaded, 18-gauge cutting needle, the Biopty gun (Bard Biopsy System, Covington, GA, USA) on consecutive patients between July 1993 and February 1995. Fifty men and 27 women were included in the study; the mean age was 45 years (range 15-69 years). The average number of punctures per patient was 5.2, with a range of 2-9, yielding an average of 4.8 samples per patient (range 1-7). The length of the samples varied from 10 to 22 mm with a constant diameter of 1 mm. The mean time required to complete the procedure was 48 min (43-52 min). Histological diagnoses were obtained in 74 of 77 patients (96%), with non-diagnostic specimens attributed to excessive fragmentation (3 cases). Complications occurred in 10 patients (puncture site hematoma, carotid artery puncture, abdominal pain, vasovagal reaction, hepatic capsule perforation, and hemobilia). The latter two complications were self-limited. In our experience this transjugular hepatic biopsy method is promising for performing biopsies in patients with chronic liver disease, due to its high success rate and low morbidity rate.
Matsuzaki, Ippei; Miyahara, Ryoji; Hirooka, Yoshiki; Funasaka, Kohei; Yamamura, Takeshi; Ohno, Eizaburo; Nakamura, Masanao; Kawashima, Hiroki; Watanabe, Osamu; Kobayashi, Makoto; Shimoyama, Yoshie; Nakamura, Shigeo; Goto, Hidemi
Background and study aims: Endoscopic tissue acquisition techniques using needle-knife and biopsy forceps allow abundant tissue acquisition from upper gastrointestinal subepithelial lesions; however, these techniques cannot capture real-time intratumor information. The aim of this study was to evaluate the feasibility of endoscopic ultrasound-guided forceps biopsy (EUS-FB) from upper gastrointestinal subepithelial lesions using a forward-viewing echoendoscope. Patients and methods: This study was a prospective case series. After mucosal cuts, several specimens were taken using a hot biopsy forceps under real-time EUS visualization. The incision was closed using hemoclips. Diagnostic yield, rate of diagnosable samples obtained under EUS visualization, procedure time, and adverse events were assessed. Results: Ten patients (median lesion size 16 mm, range 15 – 44 mm) underwent EUS-FB. The overall rate of histological diagnosis by EUS-FB was 100 % (