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At least 613 records · Page 34Linked to original sources
PubMed2000

Duplex PCR for differential identification of Mycobacterium bovis, M. avium, and M. avium subsp. paratuberculosis in formalin- fixed paraffin-embedded tissues from cattle.

We previously isolated and sequenced two genomic segments of Mycobacterium avium subsp. paratuberculosis, namely, f57, a species-specific sequence, and the p34 gene, coding for a 34-kDa antigenic protein. Comparison of sequences upstream of the p34 open reading frame (us-p34) from M. avium subsp. paratuberculosis and M. tuberculosis showed a 79-base deletion in M. tuberculosis. Sequence analysis of the p34 genes in another two species, M. bovis (strain BCG) and M. avium (strain D4), confirmed the differences observed between tuberculous and nontuberculous species. A duplex diagnostic PCR strategy based on coamplification of nonhomologous us-p34 and species-specific f57 sequences was therefore developed. Duplex PCR yielded three different patterns, specific either for tuberculous bacilli (M. tuberculosis, M. bovis, and M. africanum), for both nontuberculous mycobacteria M. avium and M. intracellulare, or for M. avium subsp. paratuberculosis. The specificity of this single-step DNA-based assay was assessed on DNA from cultured mycobacterial strains, as well as on a panel of formalin-fixed and paraffin-embedded tissues from cattle. Molecular assay results from tissular DNA were compared to conventional bacteriological and histological test results, including those obtained by Ziehl-Neelsen staining on tissue biopsy specimens. Molecular discrimination was successful and confirmed the value of duplex us-p34 and f57 sequence amplification for differential diagnosis of tuberculosis, paratuberculosis, or infections caused by other members of the M. avium complex.

Animals↗
PubMed2000

Detection of Streptococcus suis by in situ hybridization, indirect immunofluorescence, and peroxidase-antiperoxidase assays in formalin-fixed, paraffin-embedded tissue sections from pigs.

Streptococcus suis is an important pathogen in pigs and is considered a zoonotic agent. To aid diagnosis of infection caused by S. suis, a species-specific probe targeting 16S ribosomal RNA was designed and used for fluorescent in situ hybridization. Two additional immunohistochemical detection methods, an indirect immunofluorescence assay and a peroxidase-antiperoxidase method, using polyclonal antibodies also were developed. The specificity of the oligonucleotide probe was examined by whole-cell and dot-blot hybridization against reference strains of the 35 serotypes of S. suis and other closely related streptococci and other bacteria commonly isolated from pigs. The probe was specific for S. suis serotypes 1-31. The specificity of the polyclonal antibodies, which has previously been evaluated for use in diagnostic bacteriology for typing of serotype 2, was further evaluated in experimentally infected murine tissue with pure culture of different serotypes of S. suis, related streptococci, and other bacteria commonly found in pigs. The polyclonal antibodies against S. suis serotype 2 cross-reacted with serotypes 1 and 1/2 in these assays. The in situ hybridization and the immunohistochemical methods were used for detection of S. suis in formalin-fixed, paraffin-embedded tissue sections of brain, endocardium, and lung from pigs infected with S. suis. The methods developed were able to detect single cells of S. suis in situ in the respective samples, whereas no signal was observed from control tissue sections that contained organisms other than S. suis. These techniques are suitable for determining the in vivo localization of S. suis for research and diagnostic purposes.

Animals↗
PubMed1992

A rapid and simple procedure for the routine detection of ras point mutations in formalin-fixed, paraffin-embedded tissues.

The use of the polymerase chain reaction (PCR) to detect specific DNA sequences in small amounts of tissues or cells has become a widespread tool in the field of molecular biology. With the better understanding of the clinical significance of oncogene activations in human tumors, the application of PCR in a routine setting is rapidly gaining importance. We have developed a rapid and simple procedure for the detection of mutated ras oncogenes in routinely fixed, paraffin-embedded tissue samples. DNA is isolated from three 10 microns tissue sections by incubation with a nonionic detergent and proteinase K, and can be directly used for amplification by PCR. The amplified DNA fragments are then dot-blotted onto nylon membranes and are hybridized to radioactively labeled oligodeoxynucleotides, specific for each of the mutated ras sequences. After a selective washing procedure, only fully matched oligodeoxynucleotides remain bound to the membrane, thus revealing the nature of the sequences that were present in the starting material. With this method, the detection of point mutations in ras genes can be performed in a routine setting, and the results of the analyses can be available in as few as 3-4 days.

Base Sequence↗
PubMed1988

Monoclonal antibodies marking B-cell non-Hodgkin's lymphoma in paraffin-embedded tissue.

Traditional methods for the immunophenotypic analysis of the non-Hodgkin's lymphomas require fresh or snap-frozen tissue for flow cytometric or immunohistochemical studies. The monoclonal antibodies LN1, LN2, and L26 have been recently developed to recognize B-cell-specific antigens that survive routine tissue processing and paraffin embedding. In this study, the ability of these three antibodies to mark the neoplastic cells in 160 cases of paraffin-embedded non-Hodgkin's lymphoma relative to frozen section immunophenotype (42 T-cell, 118 B-cell), manner of fixation (B5 versus 10% buffered formalin), and histological subtype was examined. With B5-fixed tissue, the percentages of B-cell lymphoma marking with the antibodies were as follows: L26, 96.6%; LN1, 88.2%; LN2, 93.7%. With formalin-fixed tissue, the percentages of B-cell lymphoma reacting with the antibodies were: L26, 89.1%; LN1, 26.2%; LN2, 57.8%. Each of the antibodies marked a small percentage of paraffin-embedded T-cell lymphomas: L26, 4.7%; LN1, 4.7%; LN2, 7.1%. LN2, and to a lesser extent LN1, stained Reed-Sternberg cells, a feature not seen with L26. Nor did L26 mark nonlymphoid neoplasms, a feature previously reported with LN1 and LN2. Since a high percentage of B-cell lymphomas react with these antibodies and they are relatively specific for B-cells, they should prove highly useful for the evaluation of both diagnostic and experimental pathology specimens. L26 offers the distinct advantage of working well in both B5 and formalin-fixed tissues and seemingly not marking epithelial neoplasms.

Antibodies, Monoclonal↗
PubMed1998

[In situ PCR and PCR in situ hybridization of paraffin-embedded tissue. New diagnostic possibilities in pathology].

In situ polymerase chain reaction (PCR) and PCR in situ hybridization are new diagnostic tools in dermatopathology. These techniques can combine the sensitivity of PCR with the advantage of in situ hybridization to localize specific cellular structures. With optical control of the PCR reaction product in these techniques, false-positive results due to contamination can be minimized. Several working protocols have been established which allow detection of DNA and RNA sequences in a rapid and reproducible manner. Two different methods have been established to detect the amplification product, the indirect PCR in situ hybridization (PCRisHyb), and the direct in situ PCR (isPCR). An easy and reproducible method for PCRisHyb and isPCR in formalin-fixed and paraffin-embedded tissue is described and the two techniques are compared. Using both isPCR and PCRisHyb, the amplification product can be visualized with an optimal morphological preservation of the tissue. Indirect PCRisHyb showed a slightly higher specificity whereas direct isPCR was the quicker, easier and less expensive method.

Chickenpox↗
PubMedDate not supplied

Demonstration of Epstein-Barr viral DNA in paraffin-embedded tissues of Burkitt's lymphoma from Argentina using the polymerase chain reaction and in situ hybridization.

Burkitt's lymphoma (BL) is the most frequent non-Hodgkin's lymphoma in children in Argentina. Although epidemiologic studies have linked Epstein-Barr virus (EBV) to more than 90% of African BL cases but to only 10-20% of American and French cases, increased EBV-specific antibody titers were demonstrated in 73% of Argentinian patients with BL. To characterize the relationship between EBV and BL in Argentina, we analyzed paraffin-embedded tissues from 16 cases of BL for the presence of EBV DNA using the polymerase chain reaction (PCR) and in situ hybridization (ISH). PCR analysis showed that only 4 of 16 specimens contained the EBV BamW fragment, and these specimens were all from cases diagnosed in 1984. Results of ISH performed with a specific biotinylated DNA probe against the NotI/PstI fragment of EBV correlated with the PCR findings. EBV sequences were detected with ISH in 70-90% of the tumor cells from the 4 positive cases. These data may suggest an epidemic outbreak of EBV-related BL in 1984 superimposed on sporadic cases of BL, for which EBV may not have been an essential factor. This study also demonstrates the value of using molecular techniques on archival tissue to track epidemiologic trends.

Argentina↗
PubMed2005

Atypical Burkitt lymphoma arising from follicular lymphoma: demonstration by polymerase chain reaction following laser capture microdissection and by fluorescence in situ hybridization on paraffin-embedded tissue sections.

A 77-year-old man with disseminated lymphadenopathy was referred to our hospital. An inguinal lymph node biopsy specimen had the appearance of composite lymphoma consisting of follicular lymphoma and atypical Burkitt lymphoma. The bone marrow was involved, and chromosomal analysis of the marrow showed t(8;14) and t(14;18) in identical cells. The patient underwent systemic chemotherapy but died of lymphoma 4 months later. Polymerase chain reactions performed on cells obtained from follicular and diffuse components by laser capture microdissection showed the two components to be clonally identical. Moreover, fluorescence in situ hybridization performed on paraffin-embedded tissue sections demonstrated an immunoglobulin heavy chain (IGH)/BCL2 translocation signal in both follicular and diffuse components, but an IGH/c-MYC translocation signal in only the diffuse component. Accordingly, the atypical Burkitt lymphoma arose from the follicular lymphoma as a consequence of the additional IGH/c-MYC translocation, then involving the bone marrow.

Aged↗
PubMed1996

CD5 immunoreactivity of epithelial cells in thymic carcinoma and CASTLE using paraffin-embedded tissue.

Because the histologic features may resemble those of other mediastinal malignancies, thymic carcinoma can be difficult to diagnose, particularly if the primary site is uncertain. In an effort to facilitate this diagnosis, the authors have evaluated the use of immunohistochemistry with an antibody to CD5 (NCL-CD5). Nine thymic carcinomas, 15 thymomas, 8 lymphomas, 10 poorly differentiated lung carcinomas, 10 breast carcinomas, 1 mediastinal seminoma, and 1 thyroid carcinoma showing thymus-like differentiation (CASTLE) were studied. Four of 9 poorly differentiated carcinomas of the thymus were CD5 positive. The one CASTLE was CD5 positive. All other tumors were negative. CD5-positive lymphocytes were internal controls in every case. CD5 labels some thymic carcinomas in paraffin-embedded tissue, whereas other tumors studied were negative. CD5 immunoreactivity of CASTLE appears to support thymic derivation of this tumor.

Adult↗
PubMed1999

Detection of EWS-FLI-1 fusion in Ewing's sarcoma/peripheral primitive neuroectodermal tumor by fluorescence in situ hybridization using formalin-fixed paraffin-embedded tissue.

The balanced translocation t(11;22)(q24;q12) is specific for the Ewing's sarcoma/peripheral primitive neuroectodermal tumors (ES/PNETs) and results in the EWS/FLI-1 fusion transcript, which can be detected by reverse transcription polymerase chain reaction (RT-PCR). Recent studies also have used fluorescence in situ hybridization (FISH) to show the translocation; however, most of these have been performed on cell lines or touch preparations and short-term cultures of tumors. Moreover, the existing probes generally have shown only the break in the specific chromosomes rather than the translocation itself. We describe our findings with a new set of probes that localize to 22q12 (EWS) and 11q24 (FLI-1) and directly show the translocation as juxtaposed red-green signals on der(22) in nuclei extracted from formalin-fixed, paraffm-embedded tissues. After establishing the specificity of the probes (on metaphase spreads and interphase nuclei in two translocation-positive cell lines and normal peripheral blood lymphocytes), we evaluated 11 ES/PNETs and 10 other tumors (four alveolar rhabdomyosarcomas, three neuroblastomas, two lymphomas, one extramedullary myeloid tumor) using a two-color FISH assay. All 11 ES/PNETs showed fusion signals in 20% to 80% of evaluable nuclei. In two lymphoma cases, random overlapping signals were present in 2% and 4% of nuclei, whereas the remaining eight tumors were negative. The presence of t(11;22) was confirmed by RT-PCR in 10 of 11 ES/PNETs. We conclude that FISH analysis with this newly designed probe pair is a specific and sensitive method of detecting t(11;22) on routinely processed tissue and can be useful in the differential diagnosis of ES/PNETs from other small round blue cell tumors when only fixed tissue is available.

Adolescent↗
PubMed2006

PCR-based clonality analysis of B-cell lymphomas in paraffin-embedded tissues: diagnostic value of immunoglobulin kappa and lambda light chain gene rearrangement investigation.

Polymerase chain reaction (PCR)-based analysis, employed for detecting immunoglobulin heavy chain (IgH) gene rearrangements, has become a diagnostic tool widely used in the investigation of B-cell lymphomas, but the overall sensitivity of these methods does not exceed 80%, notably in germinal center (GC) and post-GC B-cell origin lymphomas. Many PCR strategies devised for detecting immunoglobulin light chain (IgL) gene rearrangements have been developed to enhance the clonality detection rates. However, the feasibility of these methods in routine clinical diagnosis using paraffin-embedded tissues has not yet been investigated sufficiently. We studied a large series of 108 cases of B-cell lymphomas, as well as 20 reactive lymphoid tissues using degenerate primers to amplify immunoglobulin kappa (Igkappa) and lambda (Iglambda) light chain genes. B-cell clonality was further investigated using semi-nested PCR for IgH gene rearrangements. B-cell clonality was detected in 74%, 56.5%, and 43.5% of cases using IgH, Igkappa, and Iglambda PCR, respectively. By combining these methods, the clonality detection rate increased to 93.5%. Only polyclonal patterns were noted in reactive lymphoid samples. We concluded that in addition to the established methods for IgH analysis, a PCR-based approach for IgL gene rearrangements analysis improves the clonality detection rate in over 90% of B-cell lymphoma cases using routine histological specimens with poor preservation of the genomic DNA.

Clone Cells↗
PubMed1989

New monoclonal antibodies recognizing epidermal differentiation-associated keratins in formalin-fixed, paraffin-embedded tissue. Keratin 10 expression in carcinoma of the vulva.

Two monoclonal antibodies (MAb) specific for differentiation-related epidermal keratins have been developed. They represent specific molecular probes for different stages of epidermal differentiation. Antibody DE-K10 is chain-specific for cytokeratin polypeptide no. 10 (56.5 kD) expressed in all suprabasal layers of the epidermis. Antibody DE-SCK is specific for modified stratum corneum keratins and thus represents a marker for the terminal step of epidermal differentiation. Since the epitopes identified by both antibodies are preserved in formalin-fixed, paraffin-embedded tissue sections, these antibodies can be used for retrospective studies of differentiation in various pathological processes. We have used antibody DE-K10 to study the cytokeratin 10 expression in 26 stage II or III vulvar squamous cell carcinomas. Preliminary data suggest an increased risk of recurrence in cytokeratin 10 negative tumours.

Antibodies, Monoclonal↗
PubMed2004

Detection of BCL2-IGH rearrangement on paraffin-embedded tissue sections obtained from a small submucosal tumor of the rectum in a patient with recurrent follicular lymphoma.

A 59-year-old woman was admitted to our hospital because of recurrent follicular lymphoma (FL). Colonoscopic examination revealed a rectal submucosal tumor (SMT) without any erosions and ulcers. In this patient, it was difficult to distinguish non-Hodgkin's lymphoma (NHL) invasion from other disorders of the colon including carcinoid tumor merely based on endoscopic findings. Histopathologic and immunohistochemical studies on biopsy specimens showed an infiltration of atypical lymphocytes that were positive for CD20 and BCL2 but negative for UCHL-1. Fluorescence in situ hybridization on paraffin-embedded tissue sections (T-FISH) identified a translocation of BCL2 with IGH gene. Based on these findings, the tumor was defined as an invasion of FL. T-FISH method is useful for the detection of a monoclonality of atypical lymphocytes in an SMT of the gastrointestinal tract, and particularly for the detection of chromosomal translocations specific to lymphoma subtypes.

Biopsy↗
PubMed1988

An immunohistological study of autoimmune encephalomyelitis and neuritis in the rabbit. Observations in the dorsal root ganglion using the freeze-dried paraffin-embedded tissue technique.

Previous studies of experimental autoimmune encephalomyelitis have shown that, in the central nervous system, the emigration of T-lymphocytes precedes that of mononuclear phagocytes during inflammatory lesion formation. In the present report, the formation of analogous lesions of autoimmune neuritis (EAN) was investigated in the dorsal root ganglia of rabbits immunized with homologous spinal cord in Freund's adjuvant. The relative time course of emigration of T-lymphocytes and mononuclear phagocytes into the ganglia was examined using monoclonal antibody labeling of both types of cells in serial sections of freeze-dried paraffin-embedded tissue. Results indicate that, unlike in the central nervous system, in the rabbit dorsal root ganglion T-lymphocytes and mononuclear phagocytes appear to emigrate simultaneously, as revealed by their concomitant presence in the earliest detectable lesions of EAN. It was also found that the cortical region of the rabbit dorsal root ganglion was a preferential site of EAN lesion formation, and that such lesions correlated well with the onset of clinical signs of paralysis. These results are discussed within the context of known "blood-tissue barriers" and the possible local modulation of inflammatory cell entry into regions of the nervous system.

Animals↗
PubMed2000

DNA ploidy status and proliferative activity as markers of malignant potential in Barrett's esophagus: flow cytometric study using routinely paraffin-embedded tissue.

Regular endoscopic surveillance is recommended for patients with Barrett's esophagus to detect dysplasia and to diagnose carcinoma while it is in an early and possibly treatable stage. However, there are numerous unknown aspects regarding the natural history of dysplasia in this disease, and there is still a need for more accurate markers of risk of a malignant change. The aim of this study was to investigate the usefulness of DNA flow cytometry in Barrett's esophagus to define subgroups of patients showing similar histologic findings but with a different malignancy potential. Routinely formalin-fixed and paraffin-embedded tissues of 43 patients with Barrett's esophagus were processed for flow cytometric measurements (ploidy, proliferative index) and the results were compared with the histologic evolution observed in these patients. Only in the group of patients with "indefinite" dysplasia did we find statistically significant differences between the samples from patients with and without progression to more severe lesions (mean proliferative index of stable patients: 5.2% versus 8.3% in patients with progression, p = 0.001, Student's t-test). The presence in the flow cytometric analysis of a DNA aneuploid cell line is closely related to the presence of severe histologic alterations (i.e., high-grade dysplasia: p < 0.001, Fisher's exact test). Our results suggest that this procedure is at least capable of distinguishing between a real, although incipient, neoplastic process and morphologic changes of a reactive or reparative type. The increment in the tissue proliferative index could be an indicator of an early genomic instability which, with time, will develop into lesions with a more altered DNA content: aneuploidy.

Adenocarcinoma↗
PubMed1998

Interphase chromosome painting of paraffin-embedded tissue in the differential diagnosis of possible germ cell tumors.

Germ cell tumors (GCTs) are the most frequent cancer in men aged 15 to 34 years. These tumors are highly responsive to therapy with platinum-containing regimens, and 80% of cases so treated can be considered cured. Cytogenetically, 80% of GCTs have an i(12p) regardless of tumor site or histopathology, and those that are i(12p) negative have other manifestations of 12p amplification. GCTs occasionally arise extragonadally, and such cases can be especially difficult to distinguish from poorly differentiated somatic carcinomas, a situation that poses a diagnostic and treatment dilemma We developed a technique for two-color fluorescence in situ hybridization chromosome painting on nuclei released from paraffin-embedded sections. In four tumors for which GCT was a differential diagnosis, we examined the 12p and 12q chromosome arm distributions by this technique. By use of 12p and 12q painting probes developed by microdissection, 12p and 12q were distinguished and their relative distributions evaluated. In each of the four cases, 12p regions seemed to be rearranged and over-represented relative to 12q regions. In three of the cases, an apparent i(12p) could be identified. These results support a diagnosis of GCT or GCT origin in these four cases. In tumors for which specific cytogenetic abnormalities are known, chromosome painting by fluorescence in situ hybridization using paraffin-embedded tissue is a useful technique to aid in the diagnosis of tumors that are difficult to differentiate. The patients can then be placed on treatment regimens appropriate for their specific tumor type.

Adult↗
PubMed1998

Detection of t(11;22)(q24;q12) translocation of Ewing's sarcoma in paraffin embedded tissue by nested reverse transcription-polymerase chain reaction.

Ewing's sarcoma is a poorly characterized malignant tumor with a relatively uniform histologic appearance, made up of densely packed small cells with round to oval nuclei, without distinct cell borders and without any structural differentiation. Often the diagnosis has to be made by exclusion. Recently, it has been made possible to identify characteristic chromosomal rearrangements associated with certain solid tumors. More than 85% of Ewing's sarcoma and peripheral neuroectodermal tumor present a specific t(11;22)(q24;q12) balanced translocation, resulting in the production of a novel chimerical EWS/FLI-1 message. Using oligonucleotide primers derived from EWS and FLI-1 complementary DNAs, we were able to use reverse transcription-polymerase chain reaction (RT-PCR) as a diagnostic tool. The described nested RT-PCR method as another supportive diagnostic method enables pathologists to differentiate small blue cell tumors not only to make correct diagnosis but also to investigate retrospective archival tumor samples, using formalin fixed paraffin embedded tissue as a source of RNA.

Chromosomes, Human, Pair 11↗
PubMed1989

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

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

Base Sequence↗
PubMed2002

Diagnosis of Wihipple disease by immunohistochemical analysis: a sensitive and specific method for the detection of Tropheryma whipplei (the Whipple bacillus) in paraffin-embedded tissue.

Whipple disease is a rare infection characterized clinically by diarrhea, fever, weight loss, arthralgia, malabsorption, and other systemic manifestations. The etiologic agent, Tropheryma whipplei, has been cultured only rarely. By using a polyclonal rabbit antibody produced against a cultured strain of T whipplei, tissue sections from 18 patients with Whipple disease were studied. Specimens from patients with histologic mimics and other infections served as control specimens. Immunostaining was identified in all 18 patients. Granular immunostaining was observed similar to that in periodic acid-Schiff (PAS) stains. In 2 patients, immunostaining was identified in specimens negative by H&E and PAS stains. In 4 patients studied before and after antibiotic therapy, immunostaining was retained but diminished in intensity and quantity. Immunostaining was not identified in any control specimen. Immunohistochemical analysis is a sensitive and specific method for the diagnosis of Whipple disease in paraffin-embedded tissue and may provide new opportunities to investigate the pathogenesis of the infection.

Actinobacteria↗