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At least 235 records · Page 13Linked to original sources
PubMed2001

Differentiation of porcine circovirus 1 and 2 in formalin-fixed, paraffin-wax-embedded tissues from pigs with postweaning multisystemic wasting syndrome by in-situ hybridisation.

Non-radioactive digoxigenin (DIG)-labelled probes that can differentiate porcine circovirus (PCV) 1 from PCV2 in formalin-fixed, paraffin-wax-embedded tissues by in-situ hybridisation were developed. A 349 base pair (bp) DNA fragment from open reading frame (ORF) 1 of PCV1 and a 481 bp DNA fragment from ORF2 of PCV2 generated by polymerase chain reaction (PCR) were used as PCV1 and PCV2 probes, respectively. A specific DIG-labelled PCV1 DNA probe did not hybridise with PCV2-infected PK-15 cells and vice versa. From the 40 field cases with postweaning multisystemic wasting syndrome tested by in-situ hybridisation, 30 (75 per cent) cases were PCV2-positive only and 10 (25 per cent) cases were positive for both PCV1 and PCV2. PCV1 and PCV2 DNAS were detected mainly in the macrophages of lymph nodes and spleens. Positive cells typically exhibited a dark brown to black reaction product mainly in the cytoplasm but also occasionally in the nucleus. In-situ hybridisation together with the differential probes developed in the present study represent an additional tool capable of differentiating of both types of PCV in formalin-fixed, paraffin-wax-embedded tissues.

Animals↗
PubMed1989

Immunohistochemical estrogen receptor determination in paraffin-embedded tissue. Prediction of response to hormonal treatment in advanced breast cancer.

A new immunohistochemical assay using a monoclonal estrogen receptor (ER) antibody (H222, Abbott Laboratories, North Chicago) for determination of ER in formalin-fixed paraffin-embedded tissue was applied to evaluate its clinical value in a group of 145 previously untreated patients with advanced breast cancer. Suitable histologic material was accessible in 137 of these patients, of whom 70% had ER-positive tumors. The ER-positive patients had a significantly longer median overall survival than ER-negative patients (67 versus 32 months, P much less than 0.001) and this was an effect of both a prolonged disease-free interval (27 versus 17 months, P less than 0.05) and a prolonged survival after recurrence (41 versus 15 months, P much less than 0.001). Response to endocrine therapy was obtained in 49% of the patients with ER-positive tumors and in 7% with ER-negative tumors (P much less than 0.001). Relationship between response and semiquantified individual staining features could not be established. It is concluded that ER analysis in formalin-fixed paraffin-embedded tissue offers clinically useful information for allocation of patients to endocrine therapy.

Aged↗
PubMed1981

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

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

Animals↗
PubMed1998

Improved mRNA in situ hybridization on formaldehyde-fixed and paraffin-embedded tissue using signal amplification with different haptenized tyramides.

We report an optimized in situ hybridization (ISH) protocol with a rapid signal amplification procedure based on catalyzed reporter deposition (CARD) to increase the sensitivity of non-isotopic mRNA ISH on formaldehyde-fixed and paraffin-embedded tissue. The CARD method is based on the deposition of haptenized tyramide molecules in the vicinity of hybridized probes catalyzed by horseradish peroxidase. Commercially available and newly synthesized haptenized tyramides, including digoxigenin-, biotin-, di- and trinitrophenyl- as well as fluorescein-tyramide, were compared. The haptenized tyramides were visualized using peroxidase conjugated anti-hapten antibodies followed by the diaminobenzidine reaction. As a test system, we applied digoxigenin-labeled oligonucleotides to detect insulin and vasoactive intestinal polypeptide mRNA in pancreatic endocrine tumors and liver metastases. Our results indicate that specificity, sensitivity, and applicability of oligonucleotide mRNA ISH can be significantly improved by using chemically digoxigenin-labeled oligonucleotide probes and signal amplification by CARD. Furthermore, all tested tyramides provided approximately equal amplification efficiency. In conclusion, CARD signal amplification should further promote mRNA ISH studies on paraffin-embedded tissues and allow for multiple-target nucleic acid detection in situ.

Dinitrophenols↗
PubMed1992

Immunohistological detection of human parvovirus B19 in formalin-fixed, paraffin-embedded tissues.

Human parvovirus B19 is a cause of aplastic crises in patients with haemolytic anaemias, prolonged bone marrow failure in the immunosuppressed, and fetal death secondary to non-immune hydrops. The immunohistological detection of parvovirus B19 in formalin-fixed, paraffin-embedded tissues has not previously been reported, and definitive diagnosis of infection in such specimens has relied on the use of specialized DNA hybridization and amplification techniques. A new monoclonal antibody to B19 capsid proteins, R92F6, was found to be capable of labelling infected cells in paraffin-embedded tissues from all 19 cases of parvovirus-related fetal hydrops tested, and in bone marrow from a child with congenital immunodeficiency and chronic parvovirus infection. Viral antigen was detected both in cytoplasmic and in nuclear distributions using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique without preceding proteolytic digestion. The viral epitope recognized appears to be highly conserved, as specimens were obtained over a 13-year period from widely spaced locations in the U.K. Antibody R92F6 should facilitate rapid diagnosis of parvovirus B19 infection in routinely processed and archival specimens.

Antibodies, Viral↗
PubMed1992

DNA extraction by sonication: a comparison of fresh, frozen, and paraffin-embedded tissues extracted for use in polymerase chain reaction assays.

DNA extraction from fixed tissues can be the most laborious and complex step in amplifying DNA by the polymerase chain reaction (PCR). We have previously reported a rapid and efficient method for extracting DNA by the use of sonication and glass beads. We have extended our experiences with this technique using fresh, frozen, and formalin-fixed paraffin-embedded tissues with and without the use of glass beads and report their results. Multiple tissue types were obtained at autopsy or as part of a surgical specimen. DNA was extracted from identical tissue when the sample was fresh, frozen, or formalin-fixed paraffin-embedded. Our results indicate that in most instances the sonication technique, which takes only 30 min from start to finish, can rapidly extract fresh, frozen, or formalin-fixed paraffin-embedded tissue and is superior to other rapid extraction techniques in terms of quality and quantity of DNA. It is much more rapid than those techniques that use long digestion periods. This technique will be of great value to those investigators extracting DNA for polymerase chain reaction assays.

DNA↗
PubMed1994

Nuclear accumulation of p53 protein correlates with mutations in the p53 gene on archival paraffin-embedded tissues of human breast cancer.

Fifty invasive ductal carcinomas of the breast were analyzed by immunohistochemistry, polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and direct sequencing after microdissection of conventional formalin-fixed, paraffin-embedded tissues. A highly significant association between the presence of p53 gene mutation and nuclear accumulation of p53 protein was found (P < 0.0001). Of 13 tumors that demonstrated p53 gene mutations, 11 (84.6%) showed nuclear accumulation of p53 protein. However, of 37 tumors in which gene mutations were not detected, only 5 (13.5%) showed nuclear accumulation of p53 protein. There was a statistically significant association between the nuclear accumulation of p53 protein and a higher histological grade (P < 0.001) or mitotic index (P < 0.01). In addition, gene mutations had a statistically significant association with a higher histological grade (P < 0.05) or mitotic index (P < 0.0001). Therefore, p53 abnormalities might be associated with an aggressive phenotype in breast cancer. We conclude that the immunohistochemical detection of nuclear p53 protein accumulation is highly associated with p53 gene mutations in archival paraffin-embedded tissues, and that this method is useful for rapid screening of p53 abnormalities.

Base Sequence↗
PubMed1992

Determination of estrogen receptors in paraffin-embedded tissue. Techniques and the value in breast cancer treatment.

Estrogen receptor (ER) analysis in breast cancer has been used in three clinical situations: to select patients with advanced breast cancer for hormonal therapy, as a prognostic parameter, and for selection of women with early breast cancer to adjuvant hormonal treatment. ER has traditionally been measured using labelled hormone in binding assays--often in dextran-coated charcoal assays (DCC). Monoclonal antibodies to ER has permitted development of a solid phase enzyme immunoassay (ER-EIA) used for quantitative determination of ER in tissue homogenates, and have also been used for determination of ER using an immunohistochemical assay in frozen sections (ER-ICA) or in formalin-fixed, paraffin-embedded tissue (ER-PAR). A large number of studies has compared ER-EIA with ER-DCC assays. There is a good linear correlation between the two types of assay but ER-EIA measure more ER and classify a larger fraction of tumors ER-positive than conventional ER assays. Lack of clinical data makes the significance of this uncertain. Numerous studies have reported on the correlation between ER-ICA and ER-DCC or ER-EIA. There is a good correlation among the assays on classification of ER status with a median 86% concordance, but a somewhat poorer correlation between semiquantified ER of immunohistochemical assays and ER determined by the quantitative methods (median coefficient of correlation 0.67). There is a large variation in the cut-off level for definition of ER-positive in immunohistochemical assays emphasizing the need for quality control studies. The major problem involved in ER analysis in paraffin-embedded tissue is a considerable loss of immunoreactivity compared to sections from frozen tissue. This can partly be overcome by modifications of the immunohistochemical technique using enzyme pretreatment and other amplification systems, but the sensitivity of ER-PAR remains lower than ER-ICA despite these modifications, and the ER status is less reliably determined in tumors with low ER contents (< 100 fmol). The prognostic value of ER-PAR was evaluated with a multivariate analysis. The endpoint was disease-free interval in systemically untreated patients with early breast cancer, and the variables used were: ER-DCC, ER-PAR, age, tumor size, tumor grade, and nodal status. A total of 133 patients from the Danish Breast Cancer Cooperative Group's (DBCG) 77c protocols had a complete set of variables. The analysis showed that only nodal status, ER-DCC, and tumor grade were significant and independent prognostic variables. An overview of larger multivariate studies on mainly node-negative patients failed to show independent prognostic significance of ER-DCC.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗
PubMed1984

Two new monoclonal antibodies (LN-1, LN-2) reactive in B5 formalin-fixed, paraffin-embedded tissues with follicular center and mantle zone human B lymphocytes and derived tumors.

Two monoclonal antibodies (LN-1, LN-2) reactive with B lymphocytes in B5 formalin-fixed, paraffin-embedded tissue sections have been produced by utilizing cell extracts from pokeweed mitogen-stimulated peripheral blood lymphocytes and diffuse histiocytic lymphoma SU-DHL-4 cells, respectively. Both monoclonal antibodies were initially identified by indirect immunofluorescence screening techniques on paraformaldehyde-acetone-fixed cell preparations. Specificity screens with 36 well-characterized human lymphoma and leukemia cell lines showed that both LN-1 and LN-2 stained cell lines of B cell lineage but were unreactive with those of T cell or, with one exception, myeloid derivation. Null cell acute lymphoblastic leukemia cell lines were found to be LN-2+ but LN-1-. The B cell specificity of these reagents was confirmed on 15 lymphoma and 17 leukemia biopsy specimens by using indirect immunofluorescence techniques. Immunoperoxidase staining of sections from B5-fixed, paraffin-embedded human lymphoid tissues showed that LN-1 bound to the cell membrane and cytoplasm of germinal center cells whereas LN-2 stained the nuclear membrane and cytoplasm of germinal center and mantle zone B lymphocytes as well as interfollicular histiocytes and thymic medullary dendritic cells. Both monoclonal antibodies failed to stain cortical thymocytes, lymph node T cells, and peripheral blood T and myeloid cells. Immunoperoxidase staining of 20 nonlymphoid human organs and tissues revealed that LN-1 reacted positively with red blood cell precursors of the bone marrow, ciliated epithelial cells of the bronchus, distal tubular cells of the kidney, and ductal cells from several organs including the breast and prostate. In contrast, LN-2 was unreactive with all human nonlymphoid organs and tissues including the bone marrow. Indirect immunofluorescence staining of a panel of 26 solid tumor cells lines showed that LN-1 was reactive with the majority of epithelium-derived cell lines, glioblastomas, and astrocytomas but was unreactive with neuroblastomas, small cell carcinoma of the lung, and sarcomas. LN-2 was unreactive with 25 of 26 of the solid tumor cell lines by these techniques. Immunobiochemical studies have shown that LN-1 recognizes a cell surface sialoantigen whereas LN-2 is directed against a 35,000 dalton nuclear membrane protein. Because of their high specificity for B cell tumors and their ability to stain B5-fixed, paraffin-embedded tissues, LN-1 and LN-2 are useful reagents for the diagnosis and classification of the human lymphomas and leukemias.

Antibodies, Monoclonal↗
PubMed2000

Monoclonal antibodies recognizing CD5, CD10 and CD23 in formalin-fixed, paraffin-embedded tissue: production and assessment of their value in the diagnosis of small B-cell lymphoma.

AIMS: Assessment of the expression of antigens CD5, CD10 and CD23 can be of value in the differential diagnosis of small B-cell lymphoma. Correct subclassification is important since optimal treatment regimes differ between the subtypes. The aim of this study was to generate monoclonal antibodies recognizing these antigens in paraffin-embedded tissue and to assess their efficacy using a panel of cases of small B-cell lymphoma of various subtypes. METHODS AND RESULTS: For each antibody synthetic recombinant protein and conventional murine hybridoma technology was employed. Monoclonal antibodies effective in formalin-fixed, paraffin-embedded tissue were successfully generated, designated NCL-CD5-4C7, NCL-CD10-270 and NCL-CD23-1B12, respectively. A series of 58 cases of small B-cell lymphoma including examples of each subtype (lymphocytic, follicle centre cell, mantle cell, marginal zone and lymphoplasmacytoid) was assembled and immunostaining for the respective antigens carried out using the monoclonal antibodies produced. Our results indicate that the antibodies are specific for their respective antigens and give the predicted phenotypic profile in the small B-cell lymphoma subtypes. CONCLUSIONS: These novel monoclonal antibodies may be of value in routine diagnostic practice.

Animals↗
PubMed2001

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.

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.

Adult↗
PubMed2005

Development of new rabbit monoclonal antibody to estrogen receptor: immunohistochemical assessment on formalin-fixed, paraffin-embedded tissue sections.

Evaluation of estrogen and progesterone receptors in breast cancer is widely used for the prediction of the response to endocrine therapy and as a biologic parameter closely related to disease prognosis. Immunohistochemistry is considered a specific, sensitive, and economic method for the determination of estrogen receptor/progesterone receptor status. The authors developed the first rabbit antiestrogen receptor monoclonal antibody (clone SP1) used in immunohistochemistry on formalin-fixed, paraffin-embedded tissue sections especially from breast carcinomas. This new antibody, compared with currently available antiestrogen receptor antibodies, has important advantages, including its reactivity even without heat-based antigen retrieval of fixed, embedded tissue sections in immunohistochemistry, and the predominance of nuclear immunostaining with only a very low cytoplasmic signal. A comparative study of immunohistochemistry on 61 histologic specimens from breast cancer cases showed that SP1 yields the same results as the well-known, standardized mouse monoclonal antibody to estrogen receptor (clone 1D5). Antibody affinity of SP1 is 8 times higher than that of 1D5. Thus, SP1 may prove of great value in the assessment of estrogen receptor status in human breast cancer.

Animals↗
PubMed1998

RNA quantitative analysis from fixed and paraffin-embedded tissues: membrane hybridization and capillary electrophoresis.

Fixed and paraffin-embedded tissues from pathology department archives are available for RNA expression analysis. We describe a general method for quantitation of specific RNA sequence extracted from single 6-8-micron human histological tissue sections cut from paraffin blocks. For each specific mRNA, the range of linear relationship between the log of the initial total RNA concentration and the log of the specific product after reverse transcription (RT)-PCR must be established. We usually perform RT with avian myeloblastosis virus (AMV)-RT, using specific antisense primers and a variable number of cycles of PCR amplification. The number of cycles must be adjusted within the range in which a linear relationship exists between the log of the amount of amplification product and the number of cycles. The quantity of specific product is standardized relative to beta-actin mRNA to normalize for the degree of RNA degradation, which can be quite different among samples. The amplification products were quantified by dot blot and 32P-labeled hybridization probe or by capillary electrophoresis with a laser-induced fluorescence detector. The intratest variation range was for the dot blot mean +/- 10% standard deviation (SD) and for the capillary electrophoresis mean +/- 3% SD.

Actins↗
PubMed2006

Diagnosis of clear cell sarcoma by real-time reverse transcriptase-polymerase chain reaction analysis of paraffin embedded tissues: clinicopathologic and molecular analysis of 44 patients from the French sarcoma group.

BACKGROUND: Clear cell sarcoma (CCS) is a rare tumor with a very poor prognosis that occurs predominantly in the distal extremities of young adults. Most patients bear the t(12;22) reciprocal translocation, which involves the EWS and ATF1 genes. The diagnosis of CCS usually is easy but may be challenging in unusual sites, and the detection of EWS-ATF1 fusion transcripts is helpful to rule out a metastatic melanoma. METHODS: Forty-four patients with CCS and 14 conventional melanomas were examined for the presence of EWS-ATF1 transcripts by using real-time polymerase chain reaction (PCR) analysis on paraffin embedded tissues, including frozen samples for 9 CCS samples and 9 melanoma samples. Prior to molecular analysis, the diagnosis of CCS was considered certain in 35 patients and as probable in 9 patients on the basis of location, histologic features, and immunohistochemical profile. Treatment modalities and follow-up were available for 41 patients with CCS. RESULTS: EWS-ATF1 fusion transcripts were detected in 38 paraffin embedded CCS tissues (86% of all samples; 93% of interpretable samples), 3 samples (7%) were negative, and 3 samples (7%) were considered uninterpretable. Fusion transcripts were detected in 7 of 9 samples for which the diagnosis of CCS was considered probable. EWS-ATF1 transcripts were not detected in the 14 samples of melanoma. Results from frozen tissues were concordant with those from all corresponding paraffin embedded samples. Twenty-eight of 41 patients (68%) experienced lymph node and/or distant metastasis, and the 5 year-survival rate was 44%. Mitotic index and histologic grade were predictive of survival and distant metastasis. CONCLUSIONS: The results of this study showed that the molecular detection of EWS-ATF1 fusion transcript by real-time PCR on paraffin embedded tissues is a sensitive and specific method for the diagnosis of CCS. It is an efficient tool for the diagnosis of unusual tumors, especially with regard to its distinction from melanoma. The current results also confirmed the poor prognosis for patients with this tumor type. Mitotic index and grade were predictive factors for survival and distant metastasis.

Adolescent↗
PubMed1993

Simple technique for detecting RNA viruses by PCR in single sections of wax embedded tissue.

The detection of specific RNA species in wax-embedded tissue sections using the polymerase chain reaction (PCR) means that gene expression can be studied and RNA viruses detected in stored histological tissue samples. This technique potentially allows the distribution of gene expression and viral replication to be studied in finely subdivided tissues. A technique is presented that has been used successfully to detect short RNA target sequences (130-420 bases) from proto-oncogene Abelson, human enteroviruses, and the sheep retrovirus Maedi-Visna virus using RNA PCR in single wax sections (20-30 microns). Various tissues were used which had not been deliberately prepared for this purpose. In a simple procedure hot xylene dewaxing is followed by acid phenol extraction of RNA and RNA PCR.

Abelson murine leukemia virus↗
PubMed1999

Assessment of DNA content in formalin-fixed, paraffin-embedded tissue of lung cancer by laser scanning cytometer.

A new cytometric device, a laser scanning cytometer was developed to overcome the limitations of flow cytometry (FCM) and image analyses. The purpose of this study was to develop a method that allows laser scanning cytometry (LSC) to be used for measuring the cellular DNA content of paraffin-embedded tissues. Paraffin-embedded lung cancer tissue from 30 patients was analyzed by both FCM (p-FCM) and LSC (p-LSC). In addition, touch preparations from fresh frozen tissues were prepared to provide material for LSC (f-LSC). The limits of agreement for the DNA indices (DI) measured by p-LSC and p-FCM were -0.07 to 0.07, indicating that for a given case, these methods would be expected to differ by no more than 0.07. The limits of agreement for comparisons between the other materials and methods were wider and depended upon the size of the measurements. Agreement between f-LSC and p-FCM was good for small DI values, but poor for large values. Agreement between f-LSC and p-LSC was poor for small and large DI values, but good for moderately sized values. Discordancies in DNA ploidy status between different materials and methods may have been caused by either the heterogeneity within tumors, sampling errors or differences in the interpretation of histograms. This method allows a comparison of the results of DNA analysis with histologic findings from hematoxylineosin-stained sections and the prognosis of the patients.

Aneuploidy↗
PubMed1993

Optimization of extraction and PCR amplification of RNA extracts from paraffin-embedded tissue in different fixatives.

A method was developed for fast and efficient isolation of RNA from paraffin-embedded tissue sections for subsequent PCR analysis. This method is based on the binding of RNA to acid-treated glass beads in the presence of a high molarity of guanidinium salt. It can be completed within an hour, and obviates the need for dewaxing and phenol/chloroform extractions. The effect of various fixatives and fixation times was tested and the amplification of actin mRNA fragments ranging in length from 82 to 507 bp was used to demonstrate the presence of RNA in the extracts. The method was compared to existing extraction techniques by studying the quality of the templates for reverse-transcriptase polymerase chain reaction amplification (RT-PCR), using virus-infected and mock-infected paraffin-embedded cell pellets as a model. PCR amplification of cellular and viral RNA was successful for RNA isolated by use of all extraction techniques, although the glass bead method was preferred for its simplicity and rapidity. Specimens fixed with formalin were found to be suitable for PCR, but the best results were obtained with acetone-fixed paraffin-embedded material. Dewaxing of tissue sections had no effect on the yield and quality of RNA extractions, and further purification of the extracts using gel filtration did not improve the results. After the protocols were optimized, rotavirus-infected cell pellets were used to demonstrate that extraction and amplification of dsRNA was possible. The information obtained from the studies with the model system was used for extraction of toroviral and rotaviral RNA from archival intestinal material. These data indicate that paraffin-embedded archival tissue can be used for RT-PCR analysis, adding an important technique to diagnostic pathology and retrospective studies.

Animals↗
PubMed2005

Species identification of mycobacteria in paraffin-embedded tissues: frequent detection of nontuberculous mycobacteria.

Diagnosis of infections caused by mycobacteria, especially nontuberculous mycobacteria still represents a difficult task both in microbiology and pathology. The aim of this study was to determine the frequency of mycobacterial DNA detectable by PCR in formalin-fixed paraffin-embedded tissues showing suspicious granulomatous lesions. A total of 190 archival specimens were analyzed, using a nested PCR protocol, which amplifies a fragment of the mycobacterial 65-kDa heat-shock protein gene. Restriction fragment-length polymorphisms and sequencing were utilized to further analyze the obtained PCR products. Corresponding microbiological culture results were available for 41 cases. We detected mycobacterial DNA in 119 cases (63%), of which 71 (60%) were positive for Mycobacterium tuberculosis complex DNA and 41 (34%) for DNA of nontuberculous mycobacteria. Seven cases (6%) could not be subtyped for technical reasons. The largest group of nontuberculous mycobacteria comprised 29 cases (25% of the 119 positive cases), which were assigned to Mycobacterium fortuitum complex. Mycobacterium avium-intracellulare complex was detected in eight (7%) cases, Mycobacterium gordonae in three (2.5%) and Mycobacterium rhodesiae in a single case (0.8%). All cases of Mycobacterium tuberculosis were unequivocally identified by restriction fragment-length polymorphism analysis. In contrast, sequencing provided a gain of information over restriction fragment-length polymorphism analysis in 37% of the nontuberculous mycobacteria cases (15 of 41). Alignment studies on DNA of nontuberculous mycobacteria showed frequent sequence variations, supporting the existence of sequevars. Comparison of molecular data to available results of microbiological culture assays showed a good concordance of 83%. In conclusion, amplification and sequencing of the mycobacterial 65-kDa heat-shock protein gene is an excellent tool for species identification of mycobacteria, especially nontuberculous mycobacteria, in formalin-fixed paraffin-embedded tissues.

Base Sequence↗