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PubMed1996

Detection and identification of mycobacteria in formalin-fixed, paraffin-embedded tissues by nested PCR and restriction enzyme analysis.

A novel assay based on a nested PCR and restriction enzyme analysis of the PCR products was developed for the rapid detection and identification of Mycobacterium bovis and M. avium-M. intracellulare species in formalin-fixed, paraffin-embedded tissue (PET) specimens. On the basis of the nucleotide sequence data obtained in the present study, general nested primers were constructed to amplify a 424-bp segment of the gene encoding the 65-kDa surface antigen of mycobacteria. The nested PCR assay proved to be highly sensitive, since as little as 5 to 10 fg of extracted mycobacterial DNA was detected. The safety of the assay as a routine method for the diagnosis of M. bovis and M. avium-M. intracellulare in PET specimens was provided by taking various precautions. In order to prevent false positivity, specific tools and procedures were applied. To detect false-negative results and assess the efficiency of the PCR, an internal standard molecule of amplification was constructed. The digestion of the amplicons with the restriction endonuclease Sau96-I allowed the identification of M. bovis and M. avium-M. intracellulare in a large number of clinical specimens. The present results indicate that PCR combined with an internal control of amplification and restriction enzyme analysis of the amplicons provides a rapid, sensitive, and reliable method for routine diagnostic laboratories to detect and identify M. bovis and M. avium-M. intracellulare in PET specimens.

Base Sequence↗

PubMed2004

[Diagnostic significance and clinical application of specific chimeric genes in soft tissue sarcomas by RT-PCR using paraffin-embedded tissues: a study of 103 specimens].

OBJECTIVE: To investigate the expression of the chimeric genes resulting from the specific chromosomal translocations in soft tissue sarcomas (STS) and its diagnostic significance for STS. METHODS: The variety of fusion transcripts were detected in 103 cases of STS, including 30 cases of synovial sarcoma (SS), 15 cases of rhabdomyosarcoma (RMS), 25 cases of Ewing's sarcoma/peripheral primitive neuroectodermal tumors (ES/pPNET), 12 cases of dermatofibrosarcoma protuberans (DFSP), 14 cases of aveolar soft part sarcoma (ASPS), 3 cases of leiomyosarcoma (LMS), 2 cases of malignant fibrous histocytoma (MFH), and 2 cases of fibrosarcoma (FS); and 20 cases of control tumors by reverse transcription-polymerase chain reaction (RT-PCR) using formalin fixed, paraffin embedded specimens. RESULTS: Of the 34 cases of SS 28 (93.3%) expressed SSX-SYT chimeric transcripts (14 were positive for SYT-SSX1, 9 for SYT-SSX2). Four of the six cases of alveolar RMS had a PAX3/PAX7-FKHR fusion transcript. None of the 9 cases of embryonic and polymorphic RMS expressed PAX3/PAX7-FKHR. Of the 25 cases of ES/pPNET, 19 were positive for EWS-FLI1 fusion transcript and 1 for EWS-ERG fusion transcript. COL1A1-PDGFB fusion transcript was expressed in 8 of the 12 cases (66.7%) of DFSP. Of the fourteen cases of ASPS, ten expressed ASPL-TFE3 fusion transcript. None of the 3 cases of LMS, 2 cases of MFH, 2 cases of FS, and 20 control cases contained any of the fusion transcript. CONCLUSION: Chimeric gene transcript resulting from specific chromosomal translocations is a reliable index for the molecular diagnosis of STS and RT-PCR assay for detection of specific fusion gene provides a useful tool for confirmation of the diagnosis of STS in diagnostically difficult cases and in retrospective studies.

Artificial Gene Fusion↗

PubMed1989

Localization of carbohydrates in thick and ultrathin LR white-embedded tissue sections oxidized by acetic anhydride in dimethyl sulfoxide.

The applicability of acetic anhydride (AA) in dimethyl sulfoxide (DMSO) for the oxidation of polysaccharide and their subsequent visualization with thiocarbohydrazide (TCH) and silver proteinate (SP) was evaluated on LR White-embedded thick and ultrathin liver sections. The results of these studies indicated that AA-DMSO-TCH-SP reaction is chemically specific on LR White-embedded tissues and that it offers distinct advantages for the localization of minute glycogen aggregates.

Acetates↗

PubMed1987

Monoclonal antibodies (MT1, MT2, MB1, MB2, MB3) reactive with leukocyte subsets in paraffin-embedded tissue sections.

The absence of reactivity on routinely prepared tissue sections has hampered the use of monoclonal antileukocyte antibodies in diagnostic histopathology. Here we describe five new antibodies reactive with leukocyte subsets in formaldehyde-fixed, paraffin-embedded tissue sections. Antibody MT1 is reactive with mature and immature T cells and not with mature B cells. MT2 is reactive with mature T cells and B cells, but not with immature T cells, activated T cells, and germinal center B cells. Antibody MB1 is reactive with all B cells, with about 50% of mature T cells, and not with immature T cells. MB2 is reactive with all B cells and not with T cells. However, MB2 also stains endothelial cells and several types of epithelial cells. MB3 is reactive with B cells and histiocytes, but not with T cells. The antibodies were tested on a series of lymphomas that were also immunophenotyped with a panel of well-established reagents on frozen tissue sections. The results indicate that the MB and MT antibodies are useful tools in the study of reactive and neoplastic disorders of the lymphoid system.

Antibodies, Monoclonal↗

PubMed1994

Angioimmunoblastic lymphadenopathy type of T-cell lymphoma and angioimmunoblastic lymphadenopathy: a clinicopathological and molecular biological study of 13 Chinese patients using polymerase chain reaction and paraffin-embedded tissues.

The morphological classification of angioimmunoblastic lymphadenopathy (AILD) or T-cell lymphoma of AILD-type (AILD-TCL) is still a subject of considerable difficulty and controversy. The aim of the current study was to examine the value of clinical, morphological, immunohistochemical variables in paraffin-embedded tissues in predicting the clonality of the respective lesion. Fifteen lymph node biopsies derived from 13 patients from Chengdu, China, were diagnosed as AILD or AILD-TCL and included in this study. The specimens were examined using a panel of monoclonal antibodies and a scoring system of morphological features. Clonality of the paraffin-embedded material was investigated using a novel polymerase chain reaction-technique to amplify rearranged T-cell receptor (TCR)-gamma sequences. Additional experiments were carried out to investigate the presence of clonal rearrangements of the immunoglobulin heavy chain (IgH) locus. We found clonal rearrangements of the TCR-gamma locus in 9 out of 15 lymph node biopsies. In 3 patients, the predominant cell clones carried clonal IgH and TCR-gamma rearrangements whereas 1 patient with polyclonal TCR-gamma pattern displayed IgH-monoclonality. The statistical evaluation of morphological and immunohistochemical data indicated that no single variable was able significantly to predict the clonality of the lesion. Furthermore, demonstrable clonality for the TCR-gamma or the IgH loci of a lesion did not correlate with a bad clinical course. Our data correlate with findings of other studies investigating AILD-TCL in Caucasian populations.

Adult↗

PubMed2005

Technology insight: Application of molecular techniques to formalin-fixed paraffin-embedded tissues from breast cancer.

Breast cancer is a heterogenous disease in terms of both clinical behavior and molecular characteristics. To develop prognostic and predictive markers for breast cancer, it would be useful to be able to analyze formalin-fixed paraffin-embedded tissue (FPET) collected and banked from completed clinical trials. RNAs extracted from FPETs are chemically modified and fragmented, and are therefore not ideal substrates for gene-expression profiling assays. However, methods are being developed to optimize the use of such RNAs for high-throughput gene expression profiling assays. For microarray analysis, existing methods may be adequate for fresh FPET, but they do not work well with older FPET. For older samples, real-time reverse transcription-polymerase chain reaction is the method of choice for gene-expression profiling.

Biopsy↗

PubMed1997

Cyclin D1 protein in multiple myeloma and plasmacytoma: an immunohistochemical study using fixed, paraffin-embedded tissue sections.

In this study, we analyzed 69 plasma cell neoplasms, including 54 multiple myelomas (MMs), 3 cases of plasma cell leukemia, and 12 plasmacytomas, for expression of cyclin D1 protein using an immuno-histochemical method applied to routinely fixed, paraffin-embedded tissue sections. Cyclin D1 was expressed in 18 (26%) of 69 plasma cell neoplasms, including 16 (30%) of 54 MMs and 2 (17%) of 12 plasmacytomas. Three cases of plasma cell leukemia were negative. Cyclin D1 expression correlated with the cytologic differentiation and histologic stage of MM. Twelve (75%) of 16 MMs had intermediate or immature cytologic features and were histologic Stage III. With use of a polymerase chain reaction assay, we also analyzed six cases (three cyclin D1 positive, three cyclin D1 negative) for the t(11;14); one MM carried the t(11;14) and expressed cyclin D1 protein. We conclude that cyclin D1 expression occurs in approximately one-quarter of plasma cell neoplasms and correlates with the degree of cytologic differentiation and histologic stage. The relatively high frequency of cyclin D1 expression, compared with the less than 5% incidence of the t(11;14) detected by conventional cytogenetics reported in the literature, suggests that upregulation of cyclin D1 protein might be the result of mechanisms other than the t(11;14).

Adult↗

PubMed2004

A new immunofluorostaining method using red fluorescence of PerCP on formalin-fixed paraffin-embedded tissues.

Immunofluorostaining, a versatile tissue staining method, is used in biomedical research because of its clear contrast and precise quantification of positive signals. However, its application in clinical diagnosis has been limited. A major obstacle is high fluorescent background of formalin-fixed, paraffin-embedded tissue sections (paraffin sections). On paraffin sections, strong and broad fluorescence of the section overlapping that of conventional fluorescent dyes such as fluorescein isothiocyanate (FITC) prevents detection of target immunofluorescence. To circumvent the background, we selected an albuminous dye, peridinin chlorophyll a protein (PerCP), for immunostaining of human tumor sections with tumor-reactive monoclonal antibodies. Red fluorescence of PerCP clearly distinguished the tumor region within the yellow-green autofluorescence of the section. Furthermore, it was possible to observe tissue morphology simultaneously without any counterstaining; autofluorescence served as counterstaining in this method. Digital quantification of PerCP-stained image intensity correlated (r2>0.99) well with extracted PerCP amount, indicating the usefulness of image quantification. We conclude that this new and simple immunofluorostaining method can be applied to pathological diagnosis of a wide range of conditions, including cancer.

Animals↗

PubMed1998

Detection of CD5 antigen on B cell lymphomas in fixed, paraffin embedded tissues using signal amplification by catalyzed reporter deposition.

CD5 surface antigen is expressed on some categories of B cell lymphomas. The detection of CD5 coexpression on malignant B cell infiltrates, particularly in small biopsy specimens, is useful in distinguishing between small lymphocytic lymphoma, mantle cell lymphoma, low grade marginal zone B cell lymphoma, and follicular small cleaved cell lymphoma. However, conflicting results have been reported with regard to the detection of CD5 antigen expression on B cell non-Hodgkin's lymphomas (B-NHLs) in fixed, paraffin embedded tissues using routine immunohistochemical (IHC) staining techniques. We used catalyzed reporter deposition (CARD) as a strategy to amplify the IHC signal and consequently increase the sensitivity of antigen detection. CARD improved detection of CD5 antigen without sacrificing specificity of the test. In our study, virtually all malignant B-NHLs with CD5 antigen expression showed strong immunoreactivity for a commercially available anti-CD5 monoclonal antibody using CARD, whereas the majority of the same lymphomas did not label for CD5 using routine IHC without CARD amplification. The concordance between CD5 antigen detection by immunophenotyping of fresh or frozen tissues and immunostaining with CARD amplification on paraffin fixed tissue sections was 100%. It appears that this method can be applied in the diagnostic evaluation of B-NHLs or in other situations that a weak antigen signal is present.

Biotin↗

PubMed1994

Determination of ABO genotypes with DNA extracted from formalin-fixed, paraffin-embedded tissues.

The gene encoding the specific glycosyltransferases which catalyze the conversion of the H antigen to A or B antigens shows a slight but distinct variation in its allelic nucleotide sequence and can be divided into 6 genotypes when digested with specific restriction enzymes. We extracted DNA from formalin-fixed, paraffin-embedded tissues using SDS/proteinase K treatment followed by phenol/chloroform extraction. The sequence of nucleotides for the A, B and O genes was amplified by the polymerase chain reaction (PCR). DNA fragments of 128 bp and 200 bp could be amplified in the second round of PCR, using an aliquot of the first round PCR product as template. Degraded DNA from paraffin blocks stored for up to 10.7 years could be successfully typed. The ABO genotype was deduced from the digestion patterns with an appropriate combination of restriction enzymes and was compatible with the phenotype obtained from the blood sample.

ABO Blood-Group System↗

PubMed1994

Immunohistochemical detection of p185 product, p21 product, and proliferating cell nuclear antigen (PCNA) in formalin-fixed, paraffin-embedded tissues from ovarian carcinomas. Preliminary data.

Immunohistochemical techniques for the detection of oncogene products and the assessment of cell kinetics can represent promising investigational tools in clinical oncology. In the present paper the immunohistochemical expression of p185, p21 and proliferating cell nuclear antigen (PCNA) was retrospectively assessed in formalin-fixed, paraffin-embedded tissue samples taken from 28 primary ovarian carcinomas at first surgery. Positive immunostaining for p185 was found in 0% of 6 Stage I and 23% of 22 Stage III-IV tumors. Positive immunostaining for p21 was observed in 0% of early and 41% of advanced carcinomas; this immunohistochemical finding correlated significantly with histologic grade (G3 vs G1-2 = 47% vs 9%, p = 0.042). Elevated PCNA immunoreactivity was detected in 33% of Stage I and 50% of Stage III-IV tumors. Among the 20 patients with advanced carcinoma who underwent cisplatin or carboplatin based chemotherapy followed by second-look laparotomy, the pathologic complete response (pCR) rate was 36% for patients with low PCNA expression and 0% for those with elevated PCNA expression. A tendency towards a higher pCR rate was also found for patients with negative immunostaining for p185 or for p21. The prognostic value of the immunohistochemical detection of p185, p21, and PCNA in ovarian carcinoma deserves to be further investigated.

Adenocarcinoma, Mucinous↗

PubMed1994

Monoclonal antibodies to Coxiella burnetii for antigenic detection in cell cultures and in paraffin-embedded tissues.

The authors developed monoclonal antibodies to Coxiella burnetti, the agent of Q fever. The selected monoclonal antibody, Cox1D8, did not cross-react with other bacteria and was used for early detection of C burnetti in shell vial cell cultures and for staining C burnetii in paraffin embedded tissues. Formalin or Bouin fixation did not alter the reactivity of the antigen with the antibody. This monoclonal antibody could be useful in the pathologic diagnosis of Q fever hepatitis and endocarditis.

Animals↗

PubMed1988

Evaluation of different fixatives and treatments for immunohistochemical demonstration of Coxiella burnetti in paraffin-embedded tissues.

Various fixatives and treatments such as acetone, methanol, Bouin fixative, modified Bouin fixative, 10% Formalin, modified methacarn, periodate-lysine-paraformaldehyde, acetone-methyl benzoate-xylene, and EDTA were evaluated for their effect on the immunoreactivity of Coxiella burnetii in paraffin-embedded tissues by using the avidin-biotin-peroxidase complex and the peroxidase-antiperoxidase procedure. C. burnetii antigen was shown to be present in liver, spleen, and uterus tissues of experimentally infected mice by all methods of fixation and treatment. A positive immunoreaction was seen in cytoplasmic vacuoles of macrophages, as extracellular rod-shaped organisms, and as residual particulate extra- and intracellular debris. Immunoreactivity and cellular preservation, however, varied substantially with the individual fixatives. Optimal immunostaining of C. burnetii was achieved by EDTA treatment and Bouin and acetone fixation. The avidin-biotin-peroxidase technique proved to be slightly more sensitive than the peroxidase-antiperoxidase procedure when primary antibody dilution was used as the criterion for sensitivity.

Animals↗

PubMed1997

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

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

Animals↗

PubMed2005

Gene mutation analysis in five cases of dermatofibrosarcoma protuberans using formalin-fixed, paraffin-embedded tissues.

Fusion of the collagen type I a 1 (COL1A1) gene with the platelet-derived growth factor B-chain (PDGFB) gene has been pointed out in dermatofibrosarcoma protuberans. Various exons of the COL1A1 gene have been shown to be involved in the fusion with exon 2 of the PDGFB gene. We studied the breakpoints of the COL1A1 gene using formalin-fixed, paraffin-embedded tumour specimens from five patients with dermatofibrosarcoma protuberans (three reconfirmations and two new cases). Reverse transcriptase-PCR was performed using paraffin-embedded tissues. Nucleotide sequence analysis was carried out using the PCR products to identify the breakpoints. The COL1A1-PDGFB fusion transcripts were detected from the tumour specimens. Sequence analysis revealed that the ends of exons 18, 29, 38, 42 and 44 in the COL1A1 gene were fused with the start of exon 2 in the PDGFB. This study identified a novel COL1A1 breakpoint, namely, exon 44 of the COL1A1 gene. Detection of the aberrant fusion transcript using formalin-fixed, paraffin-embedded tumour specimens is useful as a diagnostic aid for dermatofibrosarcoma protuberans in cases where fresh or frozen samples of tumour tissue are not available.

Adolescent↗

PubMed1996

Postmortem diagnosis of Factor V Leiden from paraffin wax embedded tissue.

Activated protein C resistance resulting from Factor V Leiden is an important inherited thrombophilia disorder which is found in 3.5% of people in the UK. The genetic defect can be detected using the PCR and the diagnosis can be made postmortem from paraffin wax embedded tissue. The presence of Factor V Leiden should be sought in all cases of unexplained sudden death resulting from venous thromboembolism.

Journal Article↗

PubMed1979

Application of the aluminum-formaldehyde (ALFA) histofluorescence method for demonstration of peripheral stores of catecholamines and indolamines in freeze-dried paraffin-embedded tissue, cryostat sections and whole-mounts.

This paper describes new procedures for highly sensitive visualization of monoamine stores in peripheral tissues, taking advantage of the recently introduced aluminum-catalysed formaldehyde (ALFA) reaction. The tissues are exposed to an aluminum sulphate solution (with or without formaldehyde fixation) in a perfusion and/or immersion step, followed by formaldehyde vapour treatment. Procedures are described for freeze-dried, paraffin embedded tissue, cryostat sections and whole mount preparations. For all these tissue preparations the ALFA method gives a highly sensitive and precise demonstration of catecholamine-containing neurons and 5-HT-containing cells in a variety of peripheral tissues. For freeze-dried tissue and cryostat sections the ALFA method represents an improvement in comparison with other available methods. This is particularly noticeable for the very delicate adrenergic nerves in such organs as the thyroid, ovary, pancreas and the gastrointestinal tract.

Aluminum↗

PubMed2003

In situ hybridization AT-tailing with catalyzed signal amplification for sensitive and specific in situ detection of human immunodeficiency virus-1 mRNA in formalin-fixed and paraffin-embedded tissues.

In situ hybridization is one of the most important techniques to visualize gene expression at the cellular level in various tissues. The in situ hybridization-AT tailing (ISH-AT) method uses a specially designed and synthesized oligonucleotide probe that has (AT)10 on the 3' side. This (AT)10 of the probe is elongated by DeltaTth DNA polymerase in the presence of dATP, dTTP, and labeled dUTP in the tissue after hybridization. Through this process the target is labeled with many hapten molecules. In this study, we detected human immunodeficiency virus type 1 RNA in formalin-fixed and paraffin-embedded tissues obtained from autopsied patients with acquired immunodeficiency syndrome by combining ISH-AT with the catalyzed signal amplification (CSA) system (ISH-AT-CSA), although we failed to detect signals from the same samples by conventional in situ hybridization using RNA probes (RISH) with CSA (RISH-CSA). We demonstrated that the ISH-AT-CSA method was superior to RISH-CSA in terms of both sensitivity and specificity, and that it was applicable to fluorescence in situ hybridization and double staining with immunohistochemistry for the characterization of cell phenotypes.

Adenine↗