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At least 253 records · Page 14Linked to original sources
PubMed2005

FISH is superior to PCR in detecting t(14;18)(q32;q21)-IgH/bcl-2 in follicular lymphoma using paraffin-embedded tissue samples.

Detection of t(14;18)(q32;q21)-IgH/bcl-2, which is present in 70% to 95% of follicular lymphomas (FLs), might aid in diagnosing FL. The efficacy of routine polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) techniques in detecting t(14;18) in paraffin-embedded tissue samples was compared on 5 normal tonsils and 28 FLs demonstrated to be t(14;18)+ by previous karyotyping. There was technical failure in 14 (50%) of the FLs by PCR, likely due to B-5 fixation, and 4 (14%) of FLs by FISH, likely due to advanced specimen age. In the remaining successful cases, 5 (36%) of 14 were positive by PCR and 24 (100%) of 24 were positive by FISH. All 5 normal tonsils were negative by both methods. FISH is superior to PCR for detecting t(14;18) from paraffin-embedded tissue samples because it is more sensitive and equally specific.

Chromosomes, Human, Pair 14↗
PubMed1997

A nonradioactive method of in situ hybridization that uses riboprobes and paraffin-embedded tissue and its combination with immunohistochemistry.

Current research into cytokine production in tissue sections relies on the detection of cytokine proteins using a variety of immunohistochemical methods. The disadvantages of this technique are that precise localization to a particular cell is difficult and it is uncertain whether the cells detected by this method are the origin or target of the cytokine or rather have nonspecifically absorbed the secreted cytokine. This question can be clarified using in situ hybridization, but current techniques are insensitive, poorly localizing, or time consuming. Biotin-labeled riboprobes were generated from cDNA fragments sandwiched between two RNA polymerase promoters (SP6 and T7 RNA polymerases) using a commercial riboprobe generation kit containing biotin-labeled UTP. The in situ hybridization technique was used to demonstrate cytokine mRNA in a range of tissues containing an inflammatory infiltrate and with a range of cytokine probes. This technique of in situ hybridization was combined with immunohistochemistry using an immunoalkaline phosphatase technique to show the powerful combination of these two techniques. The biotin-labeled riboprobes were sensitive enough to detect a range of cytokine mRNAs in a variety of tissue sections. The technique can be completed over a 24-h period and produces a stable color product that can be stored for long periods and can be quantitated using image analysis techniques. This technique was performed on paraffin-embedded tissue as well as cryosections and allowed for the detection of mRNA in archival tissue. It was also successfully combined with immunohistochemical techniques to determine simultaneously the localization of a cytokine product in particular cell lineages. A nonradioactive method for in situ hybridization using biotin-labeled riboprobes is described; it is capable of detecting mRNA products from a range of genes in a variety of tissue samples. An amplification step in the method enhances the sensitivity to a level that approaches that of radioactive methods, while maintaining the speed, safety, and simplicity of an immunoperoxidase detection system. The ability to use paraffin-embedded tissue with this method allows for improved tissue architecture and examination of archival tissue. These features should ensure greater use of in situ hybridization techniques in future research studies.

Arthritis, Rheumatoid↗
PubMed1984

Immunohistochemistry on semi-thin sections of hydroxypropyl methacrylate embedded tissues.

Hydroxypropyl methacrylate formulations for embedding hard or soft tissues are described. Dehydration, infiltration and embedding are carried out at 0-4 degrees C. Semi-thin serial sections may be stained by various techniques, including immunohistochemical and enzyme histochemical procedures. Lymphoid tissues and the detection of surface antigens may present some special problems, however, and these are discussed.

Acrylates↗
PubMed1991

DNA ploidy of malignant melanoma determined by image cytometry of fresh frozen and paraffin-embedded tissue.

Image analysis of DNA content was performed from single nuclei of melanoma monolayer imprints made from fresh frozen tissue of 14 patients with primary malignant melanoma and 16 patients with local recurrences at the incision site and local or distant metastases. This procedure requires fewer cells and is an advantage when the quantity of tumor available is limited, especially in thin low Breslow depth cutaneous melanomas. Image analysis allowed reproducible measurement of DNA ploidy from 100 cells. The frequency of aneuploidy was similar in primary and metastatic melanomas. Three of 3 patients with euploid primary melanomas showed no evidence of recurrences or metastases, though one died of unrelated disease with short follow-up. The 4 patients with primary melanoma who developed metastases had aneuploid primaries; two of these patients died of metastatic disease. Three of 4 patients with euploid metastatic tumors were free of disease at last follow-up, and 1 patient died with stable disease. Nine of 12 patients with aneuploid tumors died of metastatic disease. The frequency of DNA ploidy in the present image analysis study correlated with previous flow cytometry studies. In 9 patients with primary tumors with a Breslow depth greater than 0.75 mm, the DNA content was also determined in nuclei obtained from formalin-fixed paraffin-embedded tissue. The frequency of aneuploidy was higher in fresh tissue (7 of 9) as compared with paraffin-embedded tissue of the same cases (4 of 9).

Aneuploidy↗
PubMed2000

Telomerase activity and telomerase subunits gene expression patterns in neuroblastoma: a molecular and immunohistochemical study establishing prognostic tools for fresh-frozen and paraffin-embedded tissues.

PURPOSE: We have recently demonstrated that telomerase activity (TA) is an independent prognostic factor in neuroblastomas. In the present study, the prognostic impact of TA and gene expression of the three major telomerase subunits is evaluated by molecular and immunohistochemical techniques in fresh-frozen and paraffin-embedded tissues. PATIENTS AND METHODS: One hundred thirty-three neuroblastomas of all stages were analyzed for TA. The TA levels of 75 neuroblastoma cases were correlated with gene expression of telomerase subunits hTRT, human telomerase RNA (hTR), and telomerase protein 1 (TP1) by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), using an innovative approach on the LightCycler instrument (Roche Diagnostics, Mannheim, Germany). For selected cases, the applicability of RT-PCR and immunohistochemistry for hTRT expression analysis was investigated in paraffin-embedded tissues. TA and subunit expression patterns were correlated with traditional prognostic indicators and disease outcome. RESULTS: TA was present in a total of 39 (29.3%) of 133 neuroblastomas and in 31 (29.8%) of 104 initial neuroblastomas without cytotoxic pretreatment. TA was significantly correlated with both event-free and overall survival (P <.0001). Furthermore, we found a significant correlation between expression levels of TA and hTRT (P <.0001) as well as hTR (P <.001). Multivariate analysis revealed only TA and tumor stage but not serum lactate dehydrogenase, MYCN amplification, or age at diagnosis as independent prognostic factors. CONCLUSION: The significant correlation with clinical outcome strongly recommends that analysis of TA be incorporated into the clinical investigation of each individual neuroblastoma at the time of diagnosis. Because the mere presence or absence of TA without further quantification is sufficient basis for predicting disease outcome, the telomeric repeat amplification protocol assay could be complemented with but not replaced by analysis of hTRT or hTR expression.

Biomarkers, Tumor↗
PubMed1995

Detection of bcl-1 gene rearrangement and B-cell clonality in mantle cell lymphoma using formalin-fixed, paraffin-embedded tissues.

Mantle cell lymphoma (MCL) has recently emerged as a distinct clinicopathologic entity with characteristic molecular genetic features. Specifically, MCL are clonal B-cell neoplasms and often harbor bcl-1 gene rearrangements. Although this genetic profile is well documented, scant or no data are available on the molecular assessment of MCL using formalin-fixed, paraffin-embedded tissue as a sample source. The polymerase chain reaction (PCR) was employed to study bcl-1 and immunoglobulin heavy chain (IgH) gene rearrangements (B-cell clonality) using formalin-fixed tissue from 12 cases of MCL. In addition, 12 cases of low grade B-cell lymphoma and 5 cases of reactive lymphocytic hyperplasia were studied as comparison controls. A hemi-nested PCR assay was developed to identify major translocation cluster (MTC) bcl-1 gene rearrangements, whereas IgH gene rearrangements were evaluated by both a single-step and hemi-nested approach. Bcl-1 gene rearrangements were amplified in 4 of 12 (33%) MCL, but in none of the controls. With the hemi-nested approach, B-cell monoclonality was demonstrated in 11 of 12 (92%) MCL; 6 of 6 (100%) small lymphocytic lymphomas; 1 of 2 marginal zone lymphomas; 1 of 4 follicular lymphomas; and 0 of 5 reactive lymphocytic hyperplasias. When one-step PCR was used for B-cell clonality assessment, the overall detection rate was lower, specifically: 8 of 12 (67%) MCL; 4 of 6 (67%) small lymphocytic lymphomas; 1 of 2 marginal zone lymphomas; 0 of 4 follicular lymphomas; and 0 of 5 reactive lymphocytic hyperplasias were identified as monoclonal. We have demonstrated that MTC bcl-1 gene rearrangements can be amplified from formalin-fixed tissue. In addition, monoclonal B-cell populations from MCL are better amplified with a hemi-nested approach rather than a single-step PCR assay. With specialized nucleic acid isolation techniques and appropriate PCR protocol design, formalin-fixed, paraffin-embedded tissue is an adequate source of DNA for assessing MTC bcl-1 and IgH gene rearrangements.

B-Lymphocytes↗
PubMed1992

The monoclonal antibody (MAB) CD 68 allows the immunocytochemical identification of macrophages in primary and metastatic brain tumors in paraffin embedded tissues.

While using the MAB CD 68 applied to paraffin embedded tissue specimen, macrophages could be marked in 31 primary brain tumors of glial origin and in 17 metastatic tumors from which 7 were carcinomas of the lung, 5 carcinomas of the breast, 2 from the digestive tract and 3 were clear cell carcinomas of the kidney. Despite quantitative differences between the primary and metastatic tumors in all specimen examined macrophages were found. The macrophages were localized within the tumor tissue, at the periphery of the tumor and its surroundings. In some cases cells with an intensive staining reactions were visible in blood vessel walls. No correlation between the type of tumor and intensity of macrophages infiltration has been found, however there were quantitative differences between primary and metastatic tumors.

Adenocarcinoma↗
PubMedDate not supplied

Prognostic value of flow cytometric DNA analysis in non-small-cell lung cancer: rationale of sequential processing of frozen and paraffin-embedded tissue.

The objective of this study was to determine the prognostic information provided by flow cytometric DNA analysis in non-small-cell lung cancer. Lung samples of 132 consecutive patients submitted to surgery were prospectively processed. When no aneuploid populations were detected in fresh frozen samples, the process continued as a second step in paraffin-embedded tissue, consuming all the tumor available. The influence of ploidy on the postoperative outcome was studied by both a univariate and a multivariate analysis. Aneuploidy was found in 81 patients (61.4%). Fourteen patients showed no aneuploidy in fresh frozen samples; and only after further analysis in paraffin-embedded tissue was abnormal DNA detected. Overall, the 36-month survival was 69% for the diploid group and 24% for the aneuploid group (p = 0.0006). Including subjects submitted to complete tumor removal (stages I, II, and IIIA) in a multivariate analysis adjusted for TNM stage and histologic type, bearers of aneuploid tumors exhibited a higher risk of relapse (hazard ratio 2.65; CI 95% 1.5-4.66;p = 0.004) or death (hazard ratio 2.17; CI 95% 1.08-4.39;p = 0.032) than patients with diploid tumors. DNA ploidy resulted an independent prognostic factor of survival and tumor relapse in completely resected non-small-cell lung cancer. Sequential analysis of fresh and paraffin-embedded samples can help avoid the bias due to intratumoral DNA content heterogeneity. DNA ploidy could be an useful parameter in any future multifactorial analysis of outcome in such tumors.

Aneuploidy↗
PubMed1994

A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues.

Immunohistochemistry is a powerful tool for tissue diagnosis and research. Although the frozen section has remained the gold standard for this important approach to evaluation of antigens in tissues, there is widespread acknowledgment of many limitations. Routine paraffin-embedded sections ware widely used for morphological examination of tissues but are not optimal for antigen preservation. In this study, paraffin-embedded tissues fixed with a simple buffer containing zinc as the primary fixative were compared with tissues fixed with routine formalin, zinc-formalin, paraformaldehyde, ethanol, a variety of commercial (non-formalin-containing) fixatives that have been recommended for reduced toxicity and improved antigen survival, and frozen sections. Human lymphoid tissues and a group of antibodies to antigens (CD1, CD4, CD7, CD8, CD19) usually preserved only in frozen tissue were used as a model system. Fixation in a simple solution of zinc acetate and zinc chloride in a Tris-Ca acetate buffer resulted in antigen preservation comparable to that in frozen sections with antibodies to these cell surface markers. Morphological preservation was comparable to formalin-fixed sections. The work presents a new method that represents the closest approach yet to a technique that combines optimal antigenic survival with the convenience and morphological preservation of traditional formalin-fixed tissue embedded in paraffin.

Acetates↗
PubMed1997

A novel monoclonal antibody, RM-4, specifically recognizes rat macrophages and dendritic cells in formalin-fixed, paraffin-embedded tissues.

An anti-rat macrophage/dendritic cell monoclonal antibody, RM-4, was produced using a homogenate of silica-induced lung granulomas of rat as immunogen. Immunohistochemistry demonstrated that RM-4 was specific for macrophage and dendritic cell populations residing in various organs and tissues. It did not react with any cells other than macrophage/dendritic cells. In the double staining of the spleen, RM-4-positive macrophages showed wider distribution than those of the four other anti-rat macrophage monoclonal antibodies compared. The immunoreactivity of RM-4 was well preserved not only in frozen sections but also in formalin-fixed, paraffin-embedded tissues. The isotype of the monoclonal antibody was IgG1 kappa and its antigen molecular weight was 46 kDa. Immunoelectron microscopy revealed positive reaction products for RM-4 on the membrane of endosomes and lysosomes in macrophages and epidermal Langerhans cells. Reaction intensity increased after thioglycolate elicitation or endocytosis regardless of ingested materials. From these data, it is concluded that RM-4 recognizes a membrane protein of endolysomes in macrophages and dendritic cells. The antigen may play a role in endolysosomal processing. RM-4 is considered to be a useful tool not only for identifying macrophage/dendritic cells both in frozen and paraffin-embedded tissues, but also for evaluating their endolysosomal processing.

Animals↗
PubMed1993

Immunohistochemical detection of CD1A antigen in formalin-fixed and paraffin-embedded tissue sections with monoclonal antibody 010.

The immunoreactivity of a CD1a monoclonal antibody (MAb), denoted 010, was investigated by means of the streptavidin-biotin-peroxidase method in formalin-fixed and paraffin-embedded tissues from 47 cases. The samples comprised reactive lymphoid proliferations of skin, tonsil, and lymph node including dermatopathic lymphadenopathy and Langerhans' cell histiocytosis, Hodgkin's and non-Hodgkin's lymphomas, and thymomas. Interdigitating and dermal dendritic cells, veiled cells, Langerhans' cells, and also cortical thymocytes and their neoplastic counterparts displayed immunostaining with MAb 010 in paraffin sections. These results are identical to previous ones reported for other CD1a MAbs in fresh or frozen specimens. The findings suggest that the binding site of 010 is a fixation-resistant epitope of CD1a antigen which has not been previously identified.

Antibodies, Monoclonal↗
PubMed1989

Analysis of proliferative grade using anti-PCNA/cyclin monoclonal antibodies in fixed, embedded tissues. Comparison with flow cytometric analysis.

Cell kinetic information is an important adjunct to histologically-based tumor classifications. Presently, cell kinetic data can be obtained from slide-based material only with monoclonal antibodies such as Ki-67, which require the use of frozen sections and cannot be applied to archival, paraffin-embedded material. Monoclonal antibodies have recently been generated to PCNA/cyclin, a 36 kd, S-phase-associated nuclear protein. The authors investigated whether monoclonal antibody 19A2 could be used to identify proliferating cells within fixed, embedded tissue sections. Deparaffinized sections of 41 methacarn-fixed human tumors were immunostained with 19A2 using a streptavidin biotin immunoperoxidase system. A semiquantitative scoring system was used to evaluate the fraction of cells that were PCNA/cyclin-positive, and this score was compared with cell kinetic data obtained from parallel flow cytometric S-phase analysis that had been performed on fresh samples of the same tumors. While there was general agreement between the slide-based, antibody-derived and the flow cytometrically-derived cell kinetic information, some discrepancies were observed. Some of the latter represented cases in which the anti-PCNA/cyclin antibody preparations demonstrated significant heterogeneity in the numbers of proliferating cells in different regions of the tumor. In other cases, a significant fraction of the positive cells corresponded to nontumor stromal and/or inflammatory cells. In these cases, the slide-based method provided more information about the tumor cell population than did the flow cytometry data. It is concluded that semiquantitative immunocytochemical analysis with anti-PCNA/cyclin antibodies may represent a simple, reproducible, yet powerful technique for the routine analysis of cell kinetic data in alcohol-fixed, paraffin-embedded tissue by the surgical pathologist.

Antibodies, Monoclonal↗
PubMed2003

Fluorescence in situ hybridization detection of chromosome IGH/BCL2 translocations from paraffin-embedded tissue: evaluation in follicular lymphoma.

Follicular lymphoma is categorized as low-grade lymphoma because of the long median survival, but the disease is difficult to cure because of frequent recurrence. The t(14;18)(q32;q21) associated with follicular lymphoma juxtaposes a portion of BCL-2 (18q21) and IGH(14q32); the result is bcl-2 overexpression. In this study, a highly sensitive 2-color fluorescence in situ hybridization (FISH) method was used to detect t(14;18)(q32;q21) in the nuclei of paraffin-embedded tissue sections. Fourteen specimens, including 11 samples from follicular lymphoma and 3 samples from diffuse large cell lymphoma, for which results of karyotype study and paraffin-embedded tissues were available were selected for FISH study. The FISH results were compared with results of karyotype study of the lymph nodes involved in lymphoma. Among our 11 patients with the diagnosis of follicular lymphoma in whom karyotype study was performed, 8 had t(14;18)(q32;q21) in karyotype analysis, and 7 of these patients had a positive pattern in FISH analysis. In 1 case, FISH analysis was difficult because of weak signals. All 3 patients with diffuse large cell lymphoma and t(14;18)(q32;q21) in karyotype analysis had a positive pattern in FISH analysis. In 3 cases of follicular lymphoma without t(14;18)(q32;q21) in karyotype analysis, FISH did not show a positive pattern. Therefore the FISH assay in tissue was found to be very sensitive in detection of IGH/BCL2 translocation and was helpful in diagnosis of follicular lymphoma or in clarification of the cell origin of lymphoma when karyotype analysis was not available. Performing FISH on paraffin sections also is useful because we can identify cells with genetic abnormalities in the tumor and make a retrospective cytogenetic diagnosis even with old paraffin-embedded specimens.

Adult↗
PubMed1998

Incidental early detection of a splenic marginal zone lymphoma by polymerase chain reaction analysis of paraffin-embedded tissue.

Splenic marginal zone lymphoma (SMZL) most commonly presents with splenomegaly and stage IV disease. To our knowledge, there have been only two reported cases of SMZL without associated splenomegaly; one was detected incidentally after a bicycle accident. This previously reported case represented an early-stage SMZL with monoclonality confirmed by immunohistochemistry. We report a case of early-stage SMZL detected incidentally following a motor vehicle accident; monoclonality was confirmed by polymerase chain reaction analysis of paraffin-embedded tissue owing to the inability of confirmation by immunohistochemical techniques. We describe our findings and emphasize the importance of recognizing early stages of SMZL in incidental splenectomies by polymerase chain reaction analysis of paraffin-embedded tissue. Frozen tissue is not generally available in such cases.

Accidents, Traffic↗
PubMed1992

Immunostaining of formalin-fixed, paraffin-embedded tissues for malignant lymphomas.

With the advent of new monoclonal antibodies that are applicable to formalin-fixed, paraffin embedded sections, immunophenotyping is becoming increasingly important in the diagnosis and classification of lymphomas. However, multiple factors such as fixation, trypsinization and even type of antibodies used have certain effects on the final outcome of the staining procedure. In this paper we report our experience and the problems encountered in our laboratory when we first tried to establish a workable immunostaining protocol for formalin-fixed, paraffin embedded tissue sections using the immunoalkaline phosphatase technique.

Alkaline Phosphatase↗
PubMed2003

Evaluation of ethanol-fixed, paraffin-embedded tissues for proteomic applications.

We previously reported that ethanol fixation and paraffin embedding of tissues produce excellent histomorphology and good preservation of macromolecules. Here, we present a detailed evaluation of ethanol-fixed tissues for proteomic initiatives. When proteins were extracted from ethanol-fixed, paraffin-embedded prostate tissue, resolved by two-dimensional gel electrophoresis (2-DE), and stained by standard methods, several hundred protein molecules could be detected and successfully analyzed by mass spectrometry. Protein profiles obtained from ethanol-fixed tissues were highly similar to those observed from frozen tissues, in contrast to the poor protein recovery from formalin-fixed material. The protein content of specific cells that were microdissected from ethanol-fixed tissue sections using laser capture microdissection could also be successfully analyzed by 2-DE. We observed that eosin staining of tissue sections had a detrimental effect on protein separation, whereas hematoxylin staining had minimal consequence. In order to illustrate the applicability of ethanol-fixed tissues for proteomic discovery studies, we compared the protein profiles of patient-matched, normal prostatic epithelial cells and invasive adenocarcinoma cells obtained from ethanol-fixed, paraffin-embedded tissues. A number of differentially expressed proteins was discovered and identified by mass spectrometry. Immunohistochemical analyses performed on ethanol-fixed tissue sections were in agreement with the proteomic discovery findings. In light of these results, we conclude that ethanol-fixed tissues can be successfully utilized for proteomic analyses.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1998

A spin cartridge system for DNA extraction from paraffin wax embedded tissues.

A simple and efficient method of DNA extraction from paraffin wax embedded tissues using a spin cartridge system is described. Such DNAs were shown to be suitable for amplification by the polymerase chain reaction, which targeted two human papillomavirus genes and one globin fragment giving rise to products of 450, 150, and 110 base pairs, respectively. Different human tissues, stored for up to 20 years, were successfully amplified, demonstrating the usefulness of this very simple procedure for retrospective studies.

Centrifugation↗
PubMed2006

Direct quantification of gene expression in homogenates of formalin-fixed, paraffin-embedded tissues.

Formalin-fixed, paraffin-embedded (FFPE) tissues represent an important source of archival materials for gene expression profiling. We report here the development of a modified branch DNA assay that allows direct quantification of messenger RNA (mRNA) transcripts in homogenates from FFPE tissue sections without the need for RNA isolation and reverse transcription into cDNA. Formalin fixation essentially has no effect on the branch DNA assay, and RNA degradation only marginally reduces the signal by 2- to 3-fold. Under the same conditions, formalin fixation and RNA degradation greatly reduces real-time reverse transcription PCR (RT-PCR) efficiency, reducing signals by as much as 15- and 1400-fold, respectively. Although both technologies can generate biologically meaningful expression profiles from FFPE human lung tumor specimens, the branch DNA assay is more sensitive than real-time RT-PCR under the conditions tested. Our results therefore suggest that the branch DNA assay is an ideal tool for retrospective analysis of gene expression in archival tissues.

DNA↗