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PubMed2005

[Detection of cyclin D1 mRNA by reverse transcription-polymerase chain reaction in paraffin-embedded tissues and its diagnostic significance for mantle cell lymphoma].

OBJECTIVE: To investigate the feasibility of detecting cyclin D1 mRNA in paraffin-embedded tissues by reverse transcriptase polymerase chain reaction (RT-PCR) and competitive RT-PCR and its diagnostic and differential diagnostic significance for mantle cell lymphoma (MCL). METHODS: Paraffin-embedded samples of 36 cases of MCL, 71 cases of other small B-cell lymphomas and 20 cases of lymphoid reactive hyperplasia as control group were retrieved from archival materials. Cyclin D1 protein and its mRNA was detected by EnVision and RT-PCR and competitive RT-PCR in all samples. House-keeping gene PGK was choosen as internal control. RESULTS: (1) Cyclin D1 protein was expressed in 27 of the 38 MCL (71.1%). No cyclin D1 expression was found in the control group. (2) PGK was detected in 103 of the 116 cases (88.8%) and also detected in 34 of 36 MCL cases (94.7%). (3) cyclin D1 mRNA was detected in 34 nodal mantle cell lymphoma cases by RT-PCR in paraffin-embedded tissues. The positive rate of cyclin D1 mRNA was 94.4% in mantle cell lymphomas after exclusion of the 2 cases which were negative for both cyclin D1 mRNA and PGK. cyclin D1 mRNA was not detected in other nodal small B-cell lymphomas or lymphoid reactive hyperplasia, except 1 case of B-SLL. Sequencing analysis showed that sequences were identical to cyclin D1. (4) Cyclin D1 mRNA overexpression was detected in 27 cases of nodal mantle cell lymphoma by competitive RT-PCR in paraffin-embedded tissues. The positive rate of cyclin D1 mRNA overexpression was 75.0% in mantle cell lymphomas after exclusion of 2 cases which were negative for both cyclin D1 mRNA and PGK. cyclin D1 mRNA overexpression was not detected in other nodal small B-cell lymphomas or lymphoid reactive hyperplasia. CONCLUSION: RT-PCR and competitive RT-PCR detection of cyclin D1 mRNA overexpression could be used for the diagnosis and differential diagnosis of mantle cell lymphoma in paraffin-embedded blocks.

Cyclin D1↗

PubMed1990

Flow cytometric DNA content analysis in primary lung cancer: comparison of results from fresh and paraffin-embedded specimens.

Tumor DNA content was determined by flow cytometry in 24 surgically resected specimens from patients with primary lung cancer to compare results from fresh and paraffin-embedded specimens. We found a good correlation in DNA indices between fresh and paraffin-embedded specimens (r = .978), although one tumor with DNA near-diploidy was mistaken as DNA diploid in paraffin-embedded specimens. In the measurements of paraffin-embedded specimens, eight of 24 tumors were DNA multiploid and showed DNA content heterogeneity. These results indicate that DNA content analysis of paraffin-embedded specimens is helpful because it makes it easy to analyze multiple specimens from different parts of the tumor.

Adenocarcinoma↗

PubMed1989

Methodologic aspects of DNA assessment by means of image cytometry in tumors of the salivary glands. A comparison between the results obtained using sections and cytospin preparations from the same paraffin-embedded specimens.

The image cytometric nuclear DNA assessments on paraffin-embedded tissue sections and on Cytospin preparations of disaggregated specimens from the same cases were compared in 98 salivary gland tumors, including 21 acinic cell carcinomas, 29 mucoepidermoid carcinomas, 21 adenocarcinomas and 27 adenoid cystic carcinomas. The histogram type (diploid, tetraploid or aneuploid) and the number of cells with DNA values greater than 2.5c (expressed in relative units) were considered as variables in the correlation. A high correlation between the results in different specimens was found in acinic cell carcinomas, mucoepidermoid carcinomas and adenocarcinomas; the histogram type and the number of cells with DNA values greater than 2.5c were essentially the same between specimen types in these three tumor entities. The cases of adenoid cystic carcinomas showed a considerably lower degree of correlation: in 8 of the 27 cases, the Cytospin preparations yielded diploid histograms, while the tissue sections yielded aneuploid histograms. The number of cells with DNA values greater than 2.5c was notably lower in the Cytospin preparations from adenoid cystic carcinoma; the reasons for this exceptional behavior of the cells of adenoid cystic carcinoma are discussed. These findings demonstrate that paraffin-embedded specimens of different tumor entities, even from the same organ, can be affected differently by disaggregation procedures. While retrospective studies on disaggregated paraffin-embedded specimens can yield reliable results, comparative assessments using both DNA analysis techniques, as in this study, should be performed before a large number of cases is evaluated.

Adenocarcinoma↗

PubMed1998

Comparison of flow cytometric DNA content analysis in fresh and paraffin-embedded ovarian neoplasms: a prospective study.

DNA ploidy analysis was performed on both fresh and paraffin-embedded preparations from each of 54 malignant ovarian neoplasms. Aneuploidy was detected in both the fresh and the paraffin-embedded tissue in 19 out of 54 (35%) malignant cases. In addition, aneuploidy was detected exclusively in fresh tissue in seven of the malignant cases, and exclusively in paraffin-embedded tissue in one of the malignant cases, yielding a total of 27 out of 54 (50%) aneuploid cases. The correlation coefficient (r-value) for fresh and paraffin-embedded tissue ploidy analysis in the malignant specimens was 0.91. Although the frequency of recurrence was higher and overall survival lower in the malignant aneuploid specimens of both types, the combined analysis of DNA and survival rates indicated superior prognostic significance of fresh tissue. Of the seven patients in whose specimens aneuploidy was detected exclusively in fresh tissue, all died of recurrent disease during the follow-up period. Our finding indicates that data generated by flow cytometry analysis of formalin-fixed tissue should be interpreted with caution before the data can be used to draw clinical inferences.

Adult↗

PubMed1993

A method for detecting variability arising from errors in sample processing of paraffin-embedded tissue for DNA content analysis.

We present a method for controlling variability that may arise from inconsistencies in sample preparation for DNA content analysis of paraffin-embedded tissue. Human tonsil tissue obtained from routine surgical specimens was embedded in paraffin according to standard protocols. Fifty-micrometer sections were cut from the block and analyzed each day for 20 days to establish control ranges. One tonsil tissue section was processed in parallel with each run of clinical specimens. In this context, a run was defined as the simultaneous processing of 50-microns tissue sections for extraction of cell nuclei (dewaxing and rehydrating). If the tonsil G0/G1 peak coefficient of variation (CV) exceeded 2 SDs of the established mean, and optimum instrument performance and staining were verified, all samples prepared with the tonsil control were reprocessed. Instrument performance and staining were assessed by using the appropriate external controls. By using this rejection rule (12s), the frequency of sample reprocessing in our laboratory was approximately 6%. When the run was repeated and the tonsil control CV was within acceptable range, the G0/G1 peak CV of the corresponding clinical specimens improved 25% of the time. Because most investigators are willing to accept higher CVs for paraffin-embedded tissue than for fresh tissue, it is desirable to have a control to detect decreased peak resolution, resulting from errors in sample processing.

Cell Nucleus↗

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↗

PubMed2003

HOPE-fixation enables improved PCR-based detection and differentiation of Mycobacterium tuberculosis complex in paraffin-embedded tissues.

Standard PCR-based detection of mycobacterial DNA in paraffin-embedded specimens may lack sufficient sensitivity because of the degradation of nucleic acids caused by routinely used formalin fixation. Therefore, we set up an approach that aimed at improving the results by applying the novel HOPE-fixative in PCR-detection of mycobacteria in paraffin-embedded tissues. Comparison of PCR-results using DNA extracted from either HOPE- or formalin-fixed specimens in BCG-infected SCID-mice revealed a more than 100fold enhanced sensitivity for the HOPE-fixed material. Owing to the preservation of DNA from degradation in HOPE-fixed tissues, even differentiation within the M. tuberculosis complex was possible by spoligotyping. We therefore conclude that the HOPE-fixative is a useful tool for molecular pathology that enhances the sensitivity of PCR-based methods for the detection of pathogens in paraffin-embedded tissues compared to formalin-fixation. Owing to the better preserved DNA, improved differentiation of mycobacteria from archived materials is possible. These results promise new and a substantially wider range of possibilities in the field of molecular pathology.

Animals↗

PubMed1990

Feasibility of the immunohistochemical detection of endogenous steroids in paraffin-embedded ovarian tumours.

Formalin-fixed, paraffin-embedded samples from 25 ovarian granulosa cell tumours and six poorly differentiated ovarian carcinomas were examined immunohistochemically for the presence of estradiol, testosterone and progesterone. Twenty-four of the 25 granulosa cell tumours stained for estradiol predominantly in granulosa cells and also in the theca cells, but none of the carcinomas were positive. All the granulosa cell tumours and five out of six carcinomas were positive for progesterone, while 13 granulosa cell tumours and three carcinomas stained moderately positive for testosterone. No clear-cut relationship was observed between positivity for estradiol in granulosa cell tumours and occurrence of endometrial hyperplasia. In two cases of granulosa cell tumours, 1-15% cells showed ultrastructural features of steroid synthesis: i.e. moderate to abundant smooth endoplasmic reticulum and mitochondria with tubular cristae. This is in contrast with the diffuse immunohistochemical staining reaction of granulosa cell tumours with antisteroid antibodies. To evaluate the significance of immunohistochemical reactions for estradiol, four estradiol-containing tumours were tested by radioimmunoassay for the presence of estradiol before and after dehydration. As expected, treatment with organic solvents leads to a drastic reduction in estradiol content below the detection level of the assay. The results indicate that immunohistochemical staining for steroids of paraffin-embedded granulosa cell tumours is of limited value.

Adult↗

PubMed1997

Immunocytochemical detection of MART-1 in fresh and paraffin-embedded malignant melanomas.

MART-1 is a transmembrane protein that appears to be melanocyte specific. This study evaluated the use of a new monoclonal antibody, directed against MART-1, in fresh and paraffin-embedded specimens. Twenty-three paraffin-embedded tumors were evaluated for MART-1 immunoreactivity both before and after microwave-based antigen retrieval. After this comparison, 53 fine-needle aspirates from 43 patients with malignant melanoma were assessed for MART-1 immunoreactivity. Immunocytochemistry was performed on both cytospins and paraffin-embedded tissues that were pretreated with antigen retrieval. Seventeen (74%) of 23 tumors evaluated for immunoreactivity before and after antigen retrieval showed a significant increase in both staining intensity and the number of cells stained. When cytospins and antigen-retrieved cell blocks from 53 fine-needle aspirates were compared. 38 (72%) showed good correlation. In 13 (25%) of 53 tumors, MART-1 immunoreactivity was more intense in the cytospins, although the differences were marked in only two cases: Microwave-based antigen retrieval renders paraffin-embedded tissues nearly as sensitive as fresh material for use in the immunocytochemical detection of MART-1. This technique will allow the evaluation of MART-1 immunoreactivity in archival malignant melanomas.

Antibodies, Monoclonal↗

PubMed1998

Detection of TLS/FUS-CHOP fusion transcripts in myxoid and round cell liposarcomas by nested reverse transcription-polymerase chain reaction using archival paraffin-embedded tissues.

The reciprocal translocation t(12;16)(q13;p11) has been shown to be highly characteristic of myxoid and round cell subtypes of liposarcoma, and the TLS/FUS-CHOP fusion gene that resulted from the translocation is expected to be a diagnostic molecular marker of these sarcomas. In this study, we conducted a nested reverse transcription-polymerase chain reaction (RT-PCR)-based assay to detect the TLS/FUS-CHOP fusion gene transcripts using archival formalin-fixed, paraffin-embedded tumor specimens. Of 18 paraffin-embedded specimens from 16 myxoid and round cell liposarcoma cases, the fusion transcripts could be identified in 16 (89%) specimens from 15 (94%) cases. A sequence analysis using the PCR products confirmed that the detected messages were derived from either type I or type II TLS/FUS-CHOP fusion gene, the latter of which was predominant (80%). The results were consistent in primary and recurrent lesions of the same patients and in paraffin-embedded and snap-frozen samples from the same tumors. In two negative specimens, transcripts of the beta-actin gene could not be detected by RT-PCR, and intact mRNA including the fusion messages might have been degraded. No fusion transcripts were detected in snap-frozen or paraffin-embedded material of other types of tumors with myxoid morphology (seven myxoid malignant fibrous histiocytomas and four lipomas with myxoid change). These results indicate that this molecular assay can be applied to formalin-fixed, paraffin-embedded tumor tissues as a diagnostic aid for these subtypes of liposarcoma.

Adolescent↗

PubMed1992

DNA content in fresh versus paraffin-embedded tissue. Flow cytometric analysis of 100 tumors.

DNA ploidy analysis was determined on 100 consecutive tumors from a wide variety of sites using both fresh and paraffin-embedded tissue on the same specimen. The correlation coefficient (r) value between the methods was 0.85. Aneuploidy was detected by both methods in 51/100 (51%) of the cases. Fresh tissue analysis yielded 10 additional cases (overall 61% aneuploidy) not detected on corresponding paraffin-embedded sections, whereas paraffin-embedded analysis detected 4 additional cases (overall 55% aneuploidy) not revealed by fresh tissue analysis. Fresh tissue analysis produced lower coefficients of variation and resulted in a cleaner preparation with less cellular debris. Fresh tissue analysis was also superior to paraffin for the detection of hypodiploid, near-diploid and multiple peaks. Analysis of paraffin-embedded material allows examination of archival tissue and provides a more rapid means of long-term follow-up and statistical correlations for prognostic studies. Although the overall correlation of both methodologies for DNA analysis showed a minimal variation in results, in our experience fresh tissue analysis has an advantage and is preferable, when available, for ploidy analysis.

Cell Separation↗

PubMed2001

[Efficacy of direct paraffin embedding in cytological analysis of vitreous from proliferative vitreo-retinopathy (PVR)].

PURPOSE: To assess the efficacy of direct paraffin embedding in cytological analysis of vitreous samples in patients suffering from PVR, against the commonly used method for this purpose (cytospin). METHOD: Undiluted vitreous or vitreous fluids from 40 subsequent patients with PVR were collected and processed (samples collected from cassette at the end of aspiration line in final stage of vitrectomy process). 40 samples underwent cytospinning, 24 samples were paraffin embedded. Specimens were assessed by an expert ocular pathologist and tested samples were assigned to scoring and non scoring groups (where scoring means at least three cells per field in x60 magnification). RESULTS: All 24 paraffin embedding samples scored according to criteria while 36 of 40 cytospinned samples complied with scoring criteria. Differences were not statistically significant. Macrophages (87.5%) and pigmented cells (85%) were the most frequent findings. CONCLUSIONS: Direct paraffin embedding is useful for cytological studies in vitreous samples of PVR patients.

Humans↗

PubMed1980

DNA microspectrophotometric study in smear preparations from paraffin embedded tissue.

A DNA microspectrofluorimetric study on smears from cells detached from paraffin embedded tissue is presented. The cellular DNA values of these smears were compared with the cellular DNA values of fresh imprints and paraffin embedded sections obtained from the same tissue. The histograms were similar for fresh imprints and paraffin embedded smears while the DNA values in the sections were notably different. This method appears very suitable for retrospective studies of DNA using microspectrofluorimetry in routine histopathology.

DNA↗

PubMed1993

Optimization of extraction and PCR amplification of RNA extracts from paraffin-embedded tissue in different fixatives.

A method was developed for fast and efficient isolation of RNA from paraffin-embedded tissue sections for subsequent PCR analysis. This method is based on the binding of RNA to acid-treated glass beads in the presence of a high molarity of guanidinium salt. It can be completed within an hour, and obviates the need for dewaxing and phenol/chloroform extractions. The effect of various fixatives and fixation times was tested and the amplification of actin mRNA fragments ranging in length from 82 to 507 bp was used to demonstrate the presence of RNA in the extracts. The method was compared to existing extraction techniques by studying the quality of the templates for reverse-transcriptase polymerase chain reaction amplification (RT-PCR), using virus-infected and mock-infected paraffin-embedded cell pellets as a model. PCR amplification of cellular and viral RNA was successful for RNA isolated by use of all extraction techniques, although the glass bead method was preferred for its simplicity and rapidity. Specimens fixed with formalin were found to be suitable for PCR, but the best results were obtained with acetone-fixed paraffin-embedded material. Dewaxing of tissue sections had no effect on the yield and quality of RNA extractions, and further purification of the extracts using gel filtration did not improve the results. After the protocols were optimized, rotavirus-infected cell pellets were used to demonstrate that extraction and amplification of dsRNA was possible. The information obtained from the studies with the model system was used for extraction of toroviral and rotaviral RNA from archival intestinal material. These data indicate that paraffin-embedded archival tissue can be used for RT-PCR analysis, adding an important technique to diagnostic pathology and retrospective studies.

Animals↗

PubMed1992

Rapid detection and species identification of mycobacteria in paraffin-embedded tissues by polymerase chain reaction.

The sensitivity and specificity of the polymerase chain reaction (PCR) in the detection of mycobacteria in paraffin-embedded tissues and in crude lysates of mycobacterial cultures were assessed. Sections of formalin-fixed, paraffin-embedded tissues were deparaffinized and then subjected to a simple proteinase K and boiling lysis procedure. These preparations were used directly for PCR amplification of the 383 bp segment of the gene encoding the 65 kDa mycobacterial surface antigen. Crude lysates of mycobacteria were used as positive controls. The specificity of the PCR products was confirmed by Southern blot using a region-specific digoxigenin-labeled oligonucleotide probe and chemiluminescent detection. The 383 bp diagnostic fragment was visualized in 11 of 12 acid-fast bacilli (AFB) stain/culture-proven-positive blocks. Crude lysates of mycobacteria were detected to a sensitivity of approximately 80 organisms. Amplified fragments from paraffin-embedded tissues and mycobacterial cultures of M. tuberculosis, M. avium-intracellulare, and saprophytic mycobacteria were distinguished by digestion with Nar 1 restriction endonuclease. These results suggest that PCR amplification followed by restriction enzyme digestion of the PCR product is a rapid, specific, and highly sensitive technique for the detection and speciation of mycobacteria in paraffin-embedded tissues.

Base Sequence↗

PubMed1993

Flow cytometric analysis of DNA and nuclear protein in paraffin-embedded tissue.

Previously we described the simultaneous quantification of DNA and nuclear protein in unfixed tissue from solid tumors. The resultant 2 parameter flow cytometric analysis has several advantages over that of DNA alone. In this report, we describe a modification of the technique for the analysis of formalin-fixed paraffin-embedded tissue. Paraffin-embedded material was prepared by hydrating sections, incubating in 0.5% pepsin solution, washing, and resuspending in buffer containing nonionic detergent. The nuclei were then stained with fluorescein isothiocyanate and propidium iodide in the presence of ribonuclease. Several solid tumor tissue types have been analyzed, including breast, colon, kidney, and thymus. The best results were obtained when the initial pepsin treatment was for 1.5 h, instead of 0.5 h. Pepsin treatment for 1.5 h improved the CVs of both the DNA and nuclear protein parameters, and did not appear to reduce nuclear protein levels or to cause significant disintegration of nuclei. The DNA/nuclear protein histograms of unfixed and fixed, paraffin-embedded tissue were similar. Since tumor nuclei typically have higher protein levels than DNA-diploid nuclei, the technique reduces population overlapping and permits less subjective identification of DNA aneuploidy.

Aneuploidy↗

PubMedDate not supplied

Microdissection and gene rearrangement analysis of paraffin-embedded specimens of orbital malignant lymphoma.

PURPOSE: To determine whether a definite diagnosis of malignant lymphoma can be made from paraffin-embedded archived orbital specimens by gene rearrangement analysis using microdissection and polymerase chain reaction (PCR). METHODS: Specimens from four patients with histopathologically diagnosed orbital malignant lymphoma were examined. The malignant cells were microdissected off the paraffin-embedded specimens. DNA was extracted from the cells, and the immunoglobulin heavy chain ( IgH) gene was amplified by PCR. Gene rearrangements were detected by using primers for the third framework (FR3A), the second framework (FR2A), and the complementary determining region 3 (CDR3). Translocation of the B-cell lymphoma/leukemia-2 ( bcl-2) gene was also examined. RESULTS: Malignant cells were present on the slides of the paraffin-embedded specimens of three of four cases. The specimens from these three cases showed IgH rearrangements for FR3A, FR2A, and CDR3. A bcl-2-associated translocation was also detected in one case. CONCLUSIONS: Gene rearrangement analysis is applicable to paraffin-embedded archived orbital specimens to confirm a diagnosis of malignant lymphoma. The advantage of this method is that only a small specimen is needed because the detection sensitivity is high.

Complementarity Determining Regions↗

PubMed2002

DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle: heating under the influence of pH.

During the course of diagnostic surgical pathology, pathologists have established a large collection of formalin-fixed, paraffin-embedded tissues that form invaluable resources for translational studies of cancer and a variety of other diseases. Accessibility of macromolecules in the fixed tissue specimens is a critical issue as exemplified by heat-induced antigen retrieval (AR) immunohistochemical (IHC) staining. On the basis of observations that heating may also enhance in situ hybridization (ISH) and the similarity of formalin-induced chemical modifications that occur in protein and in DNA, we designed a study to examine the efficiency of DNA extraction from archival formalin-fixed, paraffin-embedded tissues using an adaptation of the basic principles of the AR technique, i.e., heating the tissue under the influence of different pH values. Archival paraffin blocks of lymph nodes, tonsil, and colon were randomly selected. Each paraffin block was prepared in 34 microtubes. For each paraffin block, one tube was used as a control sample, using a non-heating DNA extraction protocol. The other 33 tubes were tested using a heating protocol under 11 variable pH values (pH 2 to 12) under three different heating conditions (80, 100, and 120C). Evaluation of the results of DNA extraction was carried out by measuring yields by photometry and PCR amplification, as well as kinetic thermocycling (KTC)-PCR methods. In general, lower pH (acid) solutions gave inferior results to solutions at higher pH (alkaline). Heating tissues at a higher temperature and at pH 6-9 gave higher yields of DNA. There appeared to be a peak in terms of highest efficiency of extracted DNA at around pH 9. The average ratios 260:280 of extracted DNA also showed better values for samples heated at 120C. PCR products of three primers showed satisfactory results for DNA extracted from archival paraffin-embedded tissues by heating protocols at pH 6-12, with results that were comparable to the control sample subjected to the standard non-heating, enzymatic DNA extraction method. This study is the first to document the use of heating at an alkaline pH for DNA extraction from archival formalin-fixed, paraffin-embedded tissues, a recommendation based on the principles of AR for protein IHC. These findings may lead to a more effective protocol for DNA extraction from archival paraffin-embedded tissues and may also provide enhanced understanding of changes that occur during formalin-induced modification of nucleic acids.

Antigens↗