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At least 955 records · Page 53Linked to original sources
PubMed1993

Detection of human papilloma virus (HPV) infection in paraffin-embedded tissues of endometrial carcinoma.

Although human papilloma virus (HPV) associated lesions constitute a well recognized clinical entity in the female lower genital tract, namely vulva, vagina and cervix, few studies have demonstrated HPV infection in other genital sites, particularly in the ovary and uterine corpus. Recently, with the highly sensitive polymerase chain reaction (PCR) technique, HPV infections were found in an ovarian tumour and adenocarcinoma of the cervix. This prompted a retrospective analysis of HPV DNA in 22 cases of endometrial adenocarcinoma in order to investigate the possible carcinogenesis of HPV in the uterine corpus. In this study DNA extraction was performed from paraffinized cancerous tissues and the normal cervical counterpart. HPV 6, 11, 16 and 18 primers specific oligonucleotides were used in PCR to detect the presence of this oncogenic virus. HPV 16 DNA was found in 1 endometrial adenocarcinoma and 4 cervical tissues. Our result did not support the aetiological role of HPV in the carcinogenesis of endometrial carcinoma.

Adenocarcinoma↗
PubMed2001

Simultaneous evaluation of T- and B-cell clonality, t(11;14) and t(14;18), in a single reaction by a four-color multiplex polymerase chain reaction assay and automated high-resolution fragment analysis: a method for the rapid molecular diagnosis of lymphoproliferative disorders applicable to fresh frozen and formalin-fixed, paraffin-embedded tissues, blood, and bone marrow aspirates.

Current polymerase chain reaction (PCR) methods for the molecular diagnosis of B- and T-cell lymphomas by determination of clonality of immunoglobulin heavy chain (IgH) and T-cell receptor-gamma rearrangements and by detection of the chromosomal translocations t(14;18) and t(11;14), require several laborious and costly PCR assays for each of these diagnostic tests. We have developed a multiplex PCR assay for the simultaneous determination of B- and T-cell clonality and the detection of the chromosomal translocations t(14;18) and t(11;14) in a single reaction, using four-color fluorescence and automated high-resolution fragment analysis. The 26 primers combined in the multiplex PCR correspond to the sequences of >90% of the 69 variables and 6 join IgH genes and 100% of the T-cell receptor-gamma variables and join genes that could participate in the respective rearrangements. In addition, they detect the major and the minor breakpoint regions of the t(14;18) and the major breakpoint region of the t(11;14), and amplify the beta-globin gene as an internal control. The specificity of the multiplex PCR was confirmed by analysis of 39 T-cell lymphomas and 58 B-cell lymphomas, including 11 mantle cell lymphomas bearing the t(11;14) and 25 follicular lymphomas bearing the t(14;18), with known rearrangements and/or translocations. Fifteen samples of reactive lymphadenitis remained negative.

B-Lymphocytes↗
PubMed2002

Assessment of transcriptional gene activity in situ by application of HOPE-fixed, paraffin-embedded tissues.

We report the use of HOPE-fixation (HOPE = Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) for specimens utilized for in situ hybridization targeting mRNA. For this purpose, an optimized protocol was developed and repeatedly tested on HOPE-fixed lung specimens. We observed that neither pretreatment, permeabilizing the cells, nor prehybridization is necessary to generate signals. After deparaffinizing, the random primed digoxigenin-labeled probes are directly hybridized together with yeast tRNA for blocking unspecific signals. Detection was performed using anti digoxigenin antibodies conjugated with alkaline phosphatase and new-fuchsine or NBT/BCIP as substrates. The results were verified by RT-PCR and adequate negative controls. Signals for human surfactant protein-A and interferon-gamma-inducible protein-10 developed rapidly within 10 min, accompanied by high signal intensities comparable to those observed in immunohistochemistry. Signal enhancement by biotinyl-tyramide, although giving suitable results as well, did not lead to higher signal intensities, and thus was not necessary in conjunction with the probes tested so far. These experiments were performed with material stored under appropriate conditions (at +4 degrees C) up to five years. To sum up, these initial results, obtained with the novel HOPE-fixative, are promising as regards the enhancement of the capabilities of in situ hybridization in the future.

Cells, Cultured↗
PubMed1996

Single-tube protocol for the extraction of DNA or RNA from paraffin-embedded tissues using a starch-based adhesive.

We describe a method using an inexpensive craft glue to routinely isolate specific areas of tissue as small as 1 mm2 from paraffin sections. The tissue may be digested to release nucleic acid suitable for PCR or reverse transcription PCR. The use of this procedure obviates the requirement for manual microdissection or ultraviolet light irradiation. Tissue remaining on the slide can be stained and analyzed, allowing the precision of the extraction to be determined. The slide can be stored as a permanent record of the material taken for analysis.

Adhesives↗
PubMed1994

Clonality analysis of B-cell lymphoma in fresh-frozen and paraffin-embedded tissues: the effects of variable polymerase chain reaction parameters.

To investigate the sensitivity of polymerase chain reaction (PCR)-based detection of B-cell monoclonality and the effects of several variables, we analyzed 119 cases of B-cell lymphomas with proven IgH gene rearrangements, testing fresh-frozen and formalin-fixed materials in parallel. Using fresh-frozen tissue, 83 cases (70%) were positive with one-step PCR and high-stringency annealing. Two groups of false-negative cases were identified, Group I (16 cases) showing no PCR products, and Group II (20 cases) showing polyclonal smear patterns. Seminested PCR and/or lowered annealing stringency revealed nine additional positive cases in Group I but none in Group II. Amplification with bcl-2/JH primers resulted in five more positive cases. The overall positive rate in the fresh-frozen category was 81% (97 of 119). Parallel analysis was performed on formalin-fixed, paraffin-embedded material from 61 cases, and a high concordance rate (89%) was observed. The results indicated that fresh and formalin-fixed specimens are comparable in this PCR-based assay, and that the two groups of false-negative results can be accounted for by different reasons. In addition, we reviewed the current literature, discussed the diagnostic applications of this technique, and listed the core elements of a proposed PCR protocol that should be suitable for most laboratories.

Base Sequence↗
PubMed1990

Observations on the contrasting reaction of some electron dense stains applied on epoxy-embedded tissue sections.

Epon sections from glutaraldehyde-fixed rat bone marrow were treated with aqueous solutions of the following electron contrasting agents: uranyl acetate, ruthenium red, potassium permanganate, potassium dichromate, stannous chloride, palladium (II) chloride, sodium molybdate, phosphomolybdic acid, molybdenum heteropolyblue, phosphotungstic acid, iron(II)-phenanthroline, aluminium-hematoxylin, mercurochrome, cuprolinic blue, and sirius light turquoise blue. At the ultrastructural level, a high degree of electron opacity was always observed in mast cell granules and the crystalline inclusion (internum) of eosinophil granules. The chromatin revealed a somewhat lower and variable contrasting reaction, while the matrix (externum) of eosinophil granules appeared with scarce or no contrast. This pattern of electron opacity showed no correlation with the type of agent used; therefore, it can be assumed that binding processes based on the own chemical reactivity of the compounds are rather of secondary importance. The differential epoxy resin embedding of cell structures and the variable access of aqueous reagents through the non-polar plastic could be the predominant factors which account for these contrasting reactions.

Animals↗
PubMed1995

Use of PCR-PIRA for screening of a point mutation at codon 12 in K-ras oncogene obtained from paraffin embedded tissue sections.

Among various methods which have been developed for facilitating the screening of point mutations in human genomic DNA, PCR-Primer Introduced Restriction Analysis (PCR-PIRA) is of particular interest due to its practicality and short procedure allowing detection of point mutations by simple restriction enzyme digestion directly after PCR amplification. However, one limitation of PCR-PIRA method is the absence of restriction sites in the region of detection, thus creation of the recognition site in primers has been introduced. Detection of a point mutation at codon 12 in K-ras oncogene by BstNI requires one base change in the primer sequence so that only the normal but not mutant PCR product will be digested by the enzyme. However, false positive results generated from undigested normal DNA sequence are always obtained. This effect is compounded when it is used to analyse mixed cell populations in paraffin embedded section of cancer cells. Assay of a mutant band generated from normal DNA by densitometric quantitation enabled the determination of background values and thereby eliminated false positive results. Samples with higher ratios between mutant and normal bands than the background one after the first PCR-PIRA would be subjected to the second PCR-PIRA in order to confirm the results. Screening of such mutations in cervical carcinomas from paraffin embedded sections using the above criteria should reduce misinterpretation of PCR-PIRA results.

Base Sequence↗
PubMed2006

Use of a novel FISH assay on paraffin-embedded tissues as an adjunct to diagnosis of alveolar rhabdomyosarcoma.

A valuable diagnostic adjunct and important prognostic parameter in alveolar rhabdomyosarcoma (ARMS) is the identification of translocations t(2;13)(q35;q14) and t(1;13)(p36;q14), and the associated PAX3-FKHR and PAX7-FKHR fusion transcripts, respectively. Most RMS fusion gene type studies have been based on reverse transcriptase-polymerase chain reaction (RT-PCR) detection of the fusion transcript, a technique limited by RNA quality and failure of devised primer sets to detect unusual variants. As an alternative approach, we developed a fluorescence in situ hybridization (FISH) assay that can: (1) distinguish between the two most common ARMS-associated fusion genes; (2) identify potential unusual variant translocations; (3) assess histologic components in mixed alveolar/embryonal RMS; and (4) be performed on paraffinized tissue. FISH analyses of 75 specimens (40 ARMS, 16 ERMS, 8 mixed ARMS/ERMS, and 11 non-RMS tumors) using selected cosmid clone, bacterial, P1-derived, and yeast artificial chromosome probe sets were successful in all but two cases. Among specimens with informative results for both FISH and RT-PCR or standard karyotyping, PAX/FKHR classification results were concordant in 94.6% (53/56). The three discordant cases included one exhibiting a t(2;13) by FISH that was subsequently confirmed by repeat RT-PCR, a second showing a rearrangement of the PAX3 locus only (consistent with the presence of a PAX3 variant translocation), and a third revealing a t(2;13) by FISH that lacked this translocation cytogenetically. Both alveolar and embryonal components of the mixed ARMS/ERMS subtype were negative for PAX3, PAX7, and FKHR rearrangements, a surprising finding confirmed by RT-PCR and/or conventional karyotyping. These data demonstrate that FISH with newly designed probe sets is a reliable and highly specific method of detecting t(1;13) and t(2;13) in routinely processed tissue and may be useful in differentiating ARMS from other small round cell tumors. The findings also suggest that FISH may be a more sensitive assay than RT-PCR in some settings, capable of revealing variant translocations.

Biological Assay↗
PubMed1995

Human and monkey cholinergic neurons visualized in paraffin-embedded tissues by immunoreactivity for VAChT, the vesicular acetylcholine transporter.

The predicted C-terminal dodecapeptide of the human vesicular acetylcholine transporter (VAChT), deduced from the unique open reading frame of the recently cloned human VAChT cDNA, was conjugated through an N-terminal cysteine to keyhole limpet hemocyanin and used as an immunogen to generate polyclonal antihuman VAChT antibodies in rabbits. The distribution of the VAChT antigen in representative regions of the cholinergic nervous system was examined and compared to that of the acetylcholine biosynthetic enzyme choline acetyltransferase (ChAT), a specific marker for cholinergic neurons. VAChT immunoreactivity was localized in cell bodies of neurons in the basal forebrain and ventral horn of the spinal cord, regions in which major cholinergic projection systems to the cerebral cortex and to skeletal muscle, respectively, originate. The primate caudate nucleus contained numerous VAChT-positive interneurons. VAChT immunoreactivity was visualized in both cell bodies and extensive terminals in striatal interneurons, in contrast to formalin-fixed, deparaffinized sections stained for ChAT, in which cell bodies and fibers were stained but nerve terminals were less well visualized than with the VAChT antiserum. VAChT-positive nerve fibers were visualized in routinely immersion-fixed, paraffin-embedded human cerebral cortex, comparable to the density of fibers observed in perfusion-fixed Bouin's-postfixed monkey cerebral cortex. Extensive investment of virtually all principal ganglion cells of thoracic sympathetic ganglia of monkey and human with VAChT-positive nerve terminals was observed. VAChT-positive cell bodies, presumably corresponding to cholinergic sympathetic sudomotor neurons, were a significant fraction of the total principal cell population in monkey and human thoracic sympathetic ganglia.

Acetylcholine↗
PubMed1999

A reliable PCR amplification method for microdissected tumor cells obtained from paraffin-embedded tissue.

We report a reliable method for PCR (polymerase chain reaction) amplification of genomic DNA from PET. This method uses DNA extraction with the QIAquick kit and amplification with AmpliTaq Gold. Amplification of up to 959 bp from PET was achieved with this combination which exceeds the current reported upper limit of 800 bp. In summary, the gradual activation of the AmpliTaq Gold during thermal cycling allows both for higher-fidelity and higher-throughput PCR amplification from PET. The use of the QIAquick kit for DNA purification of PET is sensitive, reproducible and suitable for management of a high number of samples.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed2004

Ideal cooling process for paraffin-embedded tissues.

Back in the seventies everybody was convinced that it was no longer necessary to cool paraffinembedded tissues, because of the new advances in the production of paraffin. The reason for this assumption was the addition of plastic polymers and dimethyl sulfoxide. The quality of tissue sectioning improved because of these additives. The daily routine in the histology laboratories shows that it is impossible to produce good quality cutting without cooling. Cooling the paraffin by means of cooling plates or ice dishes creates drastic improvement; the cutting quality improves and it is much easier. Both improvements speed up the process and save time for the technicians and the physician. Long-term study indicates that not only the temperature but also the methodology of cooling and the cooling rate are playing a decisive role in the cutting quality.

Cold Temperature↗
PubMed1994

Identification of dengue virus-infected cells in paraffin-embedded tissue using in situ polymerase chain reaction and DNA hybridization.

Using tissue from suckling mice infected with Dengue virus, the following improved method for detecting Dengue viral RNA in tissue sections was devised. Reverse transcription of the viral RNA to DNA was followed by the in situ polymerase chain reaction to amplify the viral nucleic acid. This was followed by DNA hybridization with an alkaline phosphatase-labelled probe. The enzyme was then reacted with Fast red and counterstained with hematoxylin. Viral nucleic acid was readily demonstrated within glial cells and macrophages in brains of animals infected with two different serotypes of Dengue.

Animals↗
PubMed1995

Complete hydatidiform mole in twin pregnancy: differentiation from partial mole with interphase cytogenetic and DNA cytometric analyses on paraffin embedded tissues.

Six cases of hydatidiform mole associated with normal chorionic villi and a normal embryo/fetus (in five cases) were investigated with interphase cytogenetic and DNA cytometric analyses for diagnostic purposes. DNA probes specific for the pericentromeric regions of chromosomes 1 and X and for the long arm of chromosome Y were used. In four cases a dizygotic twin pregnancy could be proven. In these cases, the histologically normal chorionic villi showed an XY DNA-diploid pattern, consistent with a normal male conceptus, and the molar chorionic villi a XX pattern. In the other two cases an identical sex chromosomal pattern was found in the normal and in the molar villi (XX/XX and XY/XY respectively). In all six cases the molar placental tissues showed prominent trophoblastic hyperplasia with DNA-polyploidy, consistent with a complete hydatidiform mole. In two cases persistent gestational trophoblastic disease developed. It is emphasized that twin pregnancies composed of a normal conceptus and a complete mole have a relatively high risk for the development of persistent trophoblastic disease and therefore, should be carefully differentiated from triploid partial moles with a relatively low risk of persistent gestational trophoblastic disease. These case reports indicate that additional interphase cytogenetic and DNA cytometric analyses are useful in this differential diagnosis.

Adult↗
PubMed2002

Polymerase chain reaction in cutaneous tuberculosis: is it a reliable diagnostic method in paraffin-embedded tissues?

BACKGROUND: Most cutaneous tuberculosis lesions contain few bacilli, so identification of Mycobacterium tuberculosis in conventional laboratory tests is difficult. In vitro amplification of specific DNA sequences using polymerase chain reaction (PCR) has become a valuable tool in the rapid detection of slow-growing organisms like M. tuberculosis. AIM: To investigate the presence of M. tuberculosis DNA in cutaneous tuberculosis. METHODS: Twenty-two archival biopsy specimens diagnosed as cutaneous tuberculosis were investigated for the presence of M. tuberculosis DNA by PCR. Normal skin samples of 29 healthy patients were used as a control. RESULTS: Amplification of the M. tuberculosis DNA was observed in one of the cutaneous tuberculosis specimens and in a healthy control. CONCLUSION: Although PCR is a rapid diagnostic method, fixation procedures may decrease its sensitivity.

Adolescent↗
PubMed1997

Obtaining clone-specific primer and probe for the immunoglobulin heavy-chain gene from paraffin-embedded tissue of B-cell lymphoma: technical considerations.

The complementarity determining region (CDR) III of the immunoglobulin heavy-chain (IgH) gene is a tumor-specific marker for B-cell malignancies that has been widely exploited for the monitoring of minimal residual disease in B-precursor acute lymphocytic leukemia. There are a number of technical problems in applying the same technology for B-cell non-Hodgkin's lymphoma (B-NHL). Several procedures have been useful in overcoming these unique problems encountered in obtaining the tumor-specific sequence of the IgH-CDRIII in B-NHL, including the use of denaturing gradient gel electrophoresis or micromanipulation of tissue sections in separating the tumor-specific CDRIII products from those of contaminating normal B-lymphocytes. Minor modifications of a commercial kit greatly improve the purity of the polymerase chain reaction (PCR) products for sequencing. Modifications of the 5'-ends of the VH and IH primers, coupled with the cycle sequencing technique, make it possible to obtain unambiguous sequences on direct sequencing of short PCR products. Computer informatics and programs that facilitate the design of tumor-specific primers and probes from CDRIII sequences are described.

DNA Primers↗
PubMed2004

CD163: a specific marker of macrophages in paraffin-embedded tissue samples.

Paraffin-section immunohistochemical analysis was performed using a monoclonal antibody against CD163 to evaluate the antibody's usefulness in identifying cells of monocyte/macrophage lineage in normal and neoplastic conditions. Normal human tissue samples and samples from 211 hematopoietic disorders and 115 nonhematopoietic neoplasms were examined. The distribution of KP1 and PG-M1, monoclonal antibodies to the macrophage-associated CD68 antigen, also were evaluated for comparison. CD163 immunoreactivity was observed in resident macrophages of all normal tissue samples except splenic white pulp macrophages and germinal center tingible body macrophages. Among hematopoietic disorders and nonhematopoietic neoplasms, CD163 expression was restricted largely to cases of chronic myelomonocytic leukemia, histiocytic sarcoma, sinus histiocytosis with massive lymphadenopathy, and littoral cell angioma. Acute myeloid leukemias (AMLs) with monocytic differentiation were CD163- with the exception of 1 case of acute monoblastic leukemia. Most myeloid sarcomas also were CD163-. Compared with the CD68 antibodies, CD163 demonstrated greater specificity as a marker of disorders of monocyte/macrophage origin. However, immunohistochemical evaluation of CD163 expression does not seem to be a sensitive means of determining monocytic differentiation in AMLs in paraffin sections or establishing a diagnosis of myeloid sarcoma.

Antigens, CD↗
PubMed1996

Reproducibility of DNA ploidy and S-phase values from paraffin-embedded tissue.

OBJECTIVE: To determine the degree of variability of DNA flow cytometric results between two types of flow cytometers. STUDY DESIGN: Single-cell suspensions were made from 40 paraffin blocks of ovarian carcinoma using a modified Hedley procedure. These samples were consecutively analyzed in both Coulter's Profile II and Elite Flow cytometers. Gated and ungated data were collected and analyzed using Phoenix flow system's multicycle software. RESULTS: There were 16 (40%) DNA diploid, 23 (58%) DNA aneuploid and 1 DNA tetraploid tumor. The degree of variability in the DNA index coefficient of variation of the G0/G1 diploid peak, percentage of S phase, percentage of G2M, percentage of debris plus percentage of clumps were compared. Excellent correlations of the results were obtained in the DNA index (r = .999) and in percentage of S phase (r = .946). CONCLUSION: It is feasible to standardize variables of flow cytometric instruments to obtain reproducible results.

DNA, Neoplasm↗
PubMed1994

DNA of Mycobacterium tuberculosis in formalin-fixed, paraffin-embedded tissue in tuberculosis and sarcoidosis detected by polymerase chain reaction.

Infection with Mycobacterium tuberculosis is a major cause of death worldwide. Identification of mycobacteria in tissue sections is usually easily achieved by acid-fast stains, but this method sometimes gives unsatisfactory results. The authors therefore compared conventional staining techniques and polymerase chain reaction (PCR) for mycobacterial DNA sequences in 24 selected tissue samples from patients with tuberculosis. In all samples, either positive or negative with acid-fast stain, mycobacterial DNA fragments were detected. In addition, tissue samples from patients with clinically proven sarcoidosis were included as controls. Surprisingly, strong signals for mycobacterial DNA were found in 2 of 15 cases. Polymerase chain reaction is a useful technique in the demonstration of mycobacterial DNA fragments in patients with clinically suspected tuberculosis who have acid fast stain-negative histology. An epithelioid granulomatous reaction in the lung, negative by acid-fast stain and positive for mycobacterial DNA by PCR, however, does not permit a diagnosis of tuberculosis, because a positive result can also be obtained in cases of sarcoidosis. In some cases of sarcoidosis, the causal agent might be either cell wall defective mycobacteria or persistent intracellular DNA from mycobacteria.

Base Sequence↗