ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

A number of reports have indicated that RNA recovered from paraffin-embedded tissue can be used as a substrate in the polymerase chain reaction (PCR). Although it is established that RNA in paraffin-embedded tissue undergoes significant degradation, the specific contributions of different fixatives and fixation times to this degradation are not known. Mouse splenic tissue was harvested and fixed immediately for 2. 8, or 24 h in either formalin, Omnifix II, or Carnoy's fixative and then processed and embedded in paraffin. RNA was extracted from deparaffinized cubes of tissue using an adaptation of the technique described by Chomczynski and Sacchi. RNA was reverse transcribed using a random hexamer primed reaction. PCR amplification for cDNAs of the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase (HPRT) mRNAs was then performed. Although GAPDH amplification is used routinely on fresh and frozen tissues, we show that the presence of DNA contamination in the RNA preparations limits its usefulness in paraffin-embedded tissue. Amplifiable HPRT mRNA sequences were detected in nine of 12 samples fixed in Omnifix II. in four of 12 samples fixed in Carnoy's fixative, and in none of 12 formalin-fixed samples. Because of primer selection to preclude amplification of genomic HPRT. DNA contamination is not an issue when HPRT is amplified. Thus, HPRT represents the control system of choice for the evaluation of RNA in PET. The techniques described provide a rapid, uniform, and reproducible method of obtaining RNA from PET for molecular analysis, but they indicate limited utility for retrospective analysis of archival tissues.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

The possibility of detecting cytomegalovirus (CMV) in formalin-fixed tissues by polymerase chain reaction (PCR) was evaluated in necropsies from lung tissues in a total of 24 patients who either had received organ transplants or were immunocompromised. PCR using two different pairs of primers for amplification of the major immediate early antigen of CMV was performed on fresh tissues and tissues fixed for 24, 48, and 72 h in neutral buffered formalin and compared to immunohistochemistry (IHC) and in situ hybridization (ISH). The fresh tissues of nine patients with serological evidence for acute CMV infection were all positive for CMV by PCR. After formalin fixation, the majority of the patients failed to show distinct signals with one or both pairs of primers as measured by densitometry. In contrast to this, fresh tissues of 15 patients without signs of an acute CMV infection were found either negative or weakly positive by PCR. Using IHC or ISH, positive results were observed only in five of nine and four of nine patients with acute CMV infection, respectively. These data demonstrate that, if only formalin-fixed tissue is available, PCR for CMV detection should be performed using two pairs of primers and should be supported by IHC.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Simultaneous presence of gelatinase A (MMP-2) and MMP-2 messenger RNA (mRNA) in 30 malignant tumors with various degrees of differentiation and biological behavior was evaluated by immunohistochemistry and in situ hybridization. The series consisted of 10 gastric carcinomas. 10 colorectal carcinomas, five squamous skin carcinomas, and five basal cell skin tumors. MMP-2 was detected in all cases. MMP-2 mRNA was expressed in the stromal cells in all cases and was more marked in the less-differentiated gastric and colonic carcinomas; it was also detected in the neoplastic cells of poorly differentiated tumors, particularly in those of the signet-ring cell type, both in the colon and stomach. The study confirmed that stromal cells have a specific role in tumor invasion and suggests a direct relationship between neoplastic epithelium and stromal cells in the most aggressive varieties.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Determination of clonality in B-cell lymphomas is a useful diagnostic adjunct. In situ hybridization (ISH) for the detection of K and Λ mRN As has the potential to overcome some common specimen-related limitations in clonal assessment. Tritium-labeled antisense cRNA probes directed at conserved segments of the constant regions of the kappa and lambda mRNAs were used in an autoradiographic method to detect B-cell clonality. Using these probes, we analyzed 103 formalin-fixed, paraffin-embedded biopsy samples, and the results were subsequently compared to available immunophenotypic (all cases) and genotypic (50 cases) data. Of 103 samples, 82 (80%) had adequate RNA preservation as determined by actin RNA signals, and 73 (89%) of the 82 cases demonstrated concordant clonality assignment by both ISH and immunophenotyping. The remaining nine cases showed a specific form of discordance in that each exhibited no protein (Ig) expression but had evidence of mRNA immunoglobulin light-chain expression. Forty-five (90%) of 50 cases evaluated for immunoglobulin and T-cell receptor β-gene rearrangements demonstrated concordant results with respect to clonality assignment by ISH. Thus, ISH demonstrates adequate sensitivity with respect to traditional methods of clonality assessment. However, its practical utility awaits the development of nonradioactive detection methods with adequate sensitivity to improve turnaround time.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Clonal rearrangements of thebcl-1 andbcl-2 protoonco-genes are found in many B-lineage non-Hodgkin s lymphomas (NHL) and may play a role in their pathogenesis. We investigated rearrangements of thebcl-1 andbcl-2 protooncogenes in 13 cases of B lineage diffuse small cleaved-cell lymphoma (DSCL), and correlated the results with clinical history, immunophenotype, and outcome. Six cases showedbcl-2 rearrangements, including four patients with an antecedent follicular small cleaved-cell lymphoma (FSCL). Two patients had abcl-1 rearrangement, including one with a previous FSCL. Of the five patients who lacked detectablebcl-1 orbcl-2 rearrangements, one had an FSCL history. Similar to the lack of correlation between clinical history and genotype, there was no correlation between genotype and immunophenotype. Our results indicate that although DSCL is a morphologically uniform disease, different molecular genetic pathways are involved in its genesis. Follow-up showed four of the six DSCL patients withbcl-2 rearrangements were alive with a median survival of 56 months, whereas the median survival of the seven patients lacking abcl-2 rearrangement was 17 months and included only one survivor. Thusbcl-2 rearrangements in DSCL may define a patient subset with a more indolent genetic abnormality and prolonged survival.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

The purpose of this study was to determine the correlation between expression ofrasoncoproteins and the tumor stage or outcome of patients with gastric carcinoma. After the specificity of each anti-ra.v monoclonal antibody was confirmed by protein immunoblot analysis, immuno-histochemical assays for a common-ms antigen present in N-. Harvey- and Kirsten(K)-rasoncoproteins, as well as forK-rasspecific antigen, were performed on paraffin-embedded carcinoma tissue from 110 patients who underwent curative resection. By Western blot analysis, there was more p21 in fresh cancer specimens than in normal specimens.K-rasexpression distinguished advanced from early gastric carcinoma and correlated with depth of cancer invasion. Among the 110 patients, survival rates of those with carcinomas positive for the common-ra.? orK-rasantigens were significantly lower than of those with antigen-negative carcinomas(p< 0.05). In a multivariate analysis, nodal involvement(p= 0.002), serosal invasion(p= 0.012) andK-rasp21 expression(p= 0.044) were independently predictive of the recurrence. These results suggest thatK-rasp21 is a useful marker of tumor progression and poor prognosis after curative resection.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

The Biohit HPV Screening and Typing kits for in situ hybridization of human papillomavirus (HPV) DNA are now commercially available. The HPV Screening kit contains a cocktail of HPV probes, and the Typing kit contains separated hybridization probes for HPV 6, 11, 16, 18, 31, and 33. They were evaluated by comparison with an in situ hybridization (ISH) method, using the Pathogene HPV probes 6/11. 16/18, 31/33/51. One hundred anogenital biopsies from 78 women and 22 men were tested. Among them, 43% showed normal or inflammatory mucosa, 44%, koilocytosis or mild dysplasia, and 13%. moderate to severe dysplasia. Altogether, 60 specimens were positive with the ISH reference method: 17 with the HPV 6/11 probe, 12 with the HPV 16/18 probe, 16 with the HPV 31/33/51 probe, and 15 had mixed infections. The agreement between the Screening test and the homemade ISH is 91%. The Screening test has a sensitivity of 93% and a specificity of 87%. As for the Biohit Typing test, four false-negative samples, and partial or total discordance in nine and four samples, respectively, were observed when compared to our reference method. Thus the agreement between both typing ISH tests is 92%. The sensitivity of the Biohit Typing test is 93%, and the specificity, 91%. The sensitivity decreases to 72% when the 31 and 33 probes are evaluated separately. The Biohit Screening assay is simple, reliable, reproducible, and suitable for rapid routine screening. The Biohit Typing test allows the detection of a specific type of HPV DNA and also permits, in mixed HPV infection, definition of the type of associated HPV DNA. It might be of clinical value in the diagnosis or prognosis of HPV infection.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

In situ hybridization (ISH) provides a means for identifying viral genomes in the context of tissue pathology. We have developed a specific and sensitive ISH probe for the detection of cytomegalovirus (CMV) DNA in formalin-fixed. paraffin-embedded tissue sections. Digoxigenin-11-dUTP was incorporated into a 435-base pair fragment of the CMV Major Immediate Early (MIE) gene with use of the polymerase chain reaction (PCR). Hybridized probe was detected by reaction with antidigoxigenin antibody coupled to alkaline phosphatase and chromogenic substrates. This method has detected CMV infection in routine clinical specimens from a variety of tissue types, including colon, kidney, liver, and stomach. Infection in cells with and without characteristic inclusions is revealed with this probe. The background is so low that single infected cells are detected unambiguously. No cross-hybridization was observed with cells infected with other viruses of Herpesviridae. This approach may be useful for producing probes for the detection of other viral genomes in tissue sections.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID