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Results for “Paraffin Embedding”

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PubMed2003

PCR detection of Actinobacillus pleuropneumoniaeapxIV gene in formalin-fixed, paraffin-embedded lung tissues and comparison with in situ hybridization.

AIMS: Formalin-fixed, paraffin-embedded lung tissues from pigs experimentally infected with 12 Actinobacillus pleuropneumoniae serotypes were used to develop nested PCR for the detection of apxIV gene. METHODS AND RESULTS: The PCR results from formalin-fixed, paraffin-embedded tissues were compared with in situ hybridization. The apxIV gene was detected in formalin-fixed, paraffin-embedded lung tissues from all 39 pigs experimentally infected with 12 A. pleuropneumoniae serotypes by nested PCR. In situ hybridization produced a distinct positive signal in all 39 pigs experimentally infected with 12 A. pleuropneumoniae serotypes. Agreement rates between nested PCR and in situ hybridization were 100% for the detection of apxIV gene in formalin-fixed paraffin-embedded lung tissues. Acceptable PCR signals were detected from lung tissues fixed for periods up to 180 days. CONCLUSIONS: The apxIV gene is species-specific rather than serotype-specific and is therefore an important diagnostic marker. The nested PCR assay would be a useful method for the detection of apxIV gene to diagnose A. pleuropneumoniae infection when formalin-fixed tissues are submitted. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirmed the possibility of using formalin-fixed, paraffin-embedded tissues for the diagnosis of A. pleuropneumoniae infection in pigs.

Actinobacillus pleuropneumoniae↗

PubMed2000

Laser scanning cytometry can complement the flow cytometric DNA analysis in paraffin-embedded cancer samples: a paradigmatic case.

Archival studies on paraffin-embedded tumor samples are often complicated by difficulty obtaining a reliable diploid DNA standard. Nontumor cells, e.g., inflammatory and stromal cells, most often found interspersed among tumor cells, would represent a solution to this problem. Unfortunately, there is an inherent difficulty to positively identifying tumor cells in paraffin-embedded specimens. Using an aneuploid paraffin-embedded breast cancer sample, we show here that laser scanning cytometer (LSC) in conjunction with flow cytometry can help to address this issue. Following standard protocols, the tissue was deparaffinized and rehydrated, and the nuclei mechanically isolated before being exposed to propidium iodide. An aliquot served for single-parameter flow cytometric analysis, and the remaining cells were cytocentrifuged onto a microscope slide and LSC analysis was performed. The DNA histogram profiles generated by the two approaches were comparable and both showed the presence of cell populations with different DNA content. To assess the nature of these subsets, we performed a correlated measurement of DNA content and chromatin organization at the single-cell level by LSC. This allowed the identification of several subsets of nuclei. Slides were then stained with Giemsa and the nature of these subsets was assessed morphologically by exploiting the relocating capability of LSC. Inflammatory and stromal cells, residual diploid epithelial cells, and hyperdiploid tumor cells-each characterized by a peculiar coordinate pattern of DNA content and chromatin organization-could be positively identified. Diploid, nontumor cells can then be used as an internal standard for DNA ploidy.

Breast Neoplasms↗

PubMed1992

Comparison of cytofluorometric nuclear DNA ploidy analysis on fresh and paraffin embedded specimen.

The cytofluorometric nuclear DNA ploidy analysis was performed in gallbladder (GB) epithelium of eight patients of chronic cholecystitis to make a comparison between fresh and paraffin embedded tissue analysis, to make an ideal single cellular suspension, to evaluate the ploidy pattern affected by inflammation and to assess the histological correlations. Six (75%) fresh specimens and eight (100%) paraffin embedded specimens showed diploidy pattern of histogram. Two (25%) fresh specimens showed low ploidy pattern of histogram. There was a good correlation between histological findings and the ploidy patterns of 100% paraffin embedded specimens and 75% fresh specimens. The relative fluorescence intensity was found higher and more stable in paraffin embedded tissues than fresh tissues. Inflammation did not affect the ploidy patterns of paraffin embedded specimens whereas the ploidy patterns of fresh specimens were affected. It can be concluded that the DNA ploidy patterns and histological findings have a good correlation and inflammation does not affect the cytofluorometric DNA ploidy pattern when using the paraffin embedded specimens. Paraffin embedded specimens are superior to fresh specimens for making an ideal single cellular suspension to obtain stable, high, and accurate fluorescence intensity with less background fluorescence which makes the ploidy pattern behave like exact behavior of tissues.

Cholecystitis↗

PubMed1990

Detection of human papillomavirus DNA in squamous bronchial metaplasia and squamous cell carcinomas of the lung by in situ hybridization using biotinylated probes in paraffin-embedded specimens.

We examined a series of paraffin-embedded tissue specimens from 10 cases of squamous bronchial metaplasia and 33 cases of squamous cell carcinoma of the lung for histologic characteristics and for the presence and typing of human papillomavirus (HPV) by molecular in situ hybridization with biotinylated probes types 6, 11, 16 and 18 under stringent conditions (temperature, 19 degrees C). Fourteen of these lesions (32.5%) showed typical condylomatous histologic changes. Human papillomavirus DNA was present in seven (16%) specimens. Type 6 HPV DNA was detected in one of the squamous bronchial metaplasia cases. In six of the squamous cell carcinomas cases (18%), HPV DNA was identified (type 18, three cases; type 16, one case; type 11, one case; and type 6, one case); one of the squamous cell carcinoma specimens contained both HPV types 16 and 18. Our data confirm the presence of HPV DNA in squamous metaplastic bronchial mucosa and epidermoid lung carcinoma on paraffin-embedded tissues. This suggests that an HPV infection with benign or potentially oncogenic HPV types could be associated not only with genital tumors, but also with bronchial and lung tumors. The role of HPV DNA in the process of malignancy conversion is not yet known; HPV DNA could possibly be a cocarcinogenic factor. In situ hybridization with biotinylated probes is a useful and appropriate method of retrospective analysis of HPV DNA sequences in routinely paraffin-embedded lesions. It may be used to identify patients at risk of more serious or possibly malignant progression.

Biotin↗

PubMed1985

Flow cytometric determination of DNA ploidy level in nuclei isolated from paraffin-embedded tissue.

Nuclei, isolated from paraffin-embedded tissue, were stained with propidium iodide (PI) and found suitable for DNA analysis by flow cytometry (FCM). DNA-derived fluorescence intensity, however, was always decreased and had a much higher intersample variability as compared to results obtained with fresh material. Using chicken red blood cells (CRBC) as a model system, we found the lower fluorescence intensity to be due to the formalin fixation step in tissue processing. The intersample variability was found to be at least partly caused by variations in the duration of fixation. Overnight trypsinization improved the fluorescence intensity but did not reduce the intersample variability. Under all conditions tested PI binding to CRBC appeared to be saturable. Since fresh diploid or red blood cells could not be used to standardize DNA histograms, an alternative approach was developed in which nuclei from paraffin-embedded normal and tumor tissue of the same specimen were mixed. With this method DNA indices (DI) of 24 colorectal cancers were found to be closely correlated (r = 0.9877, P less than 0.001) with DI obtained with fresh tumor tissue from the same patients. The correlation of the percentages of S-phase nuclei between paraffin-extracted and fresh samples (r = 0.5875, P less than 0.05) was as high as could be expected, taking sampling differences into account. This method is an important tool for the retrospective analysis of FCM-derived DNA parameters in relation to diagnosis and prognosis of neoplasms.

Animals↗

PubMed1987

Utilization of monoclonal antibody L26 in the identification and confirmation of B-cell lymphomas. A sensitive and specific marker applicable to formalin-and B5-fixed, paraffin-embedded tissues.

Immunophenotypic analysis of paraffin-embedded tissues of lymphoproliferative disorders has been facilitated by recent developments of monoclonal antibodies that react with epitopes that survive histologic processing. Leukocyte common antigen (LCA) antibody has made a significant contribution to the immunocytochemical separation of non-Hodgkin's lymphomas from nonlymphoid neoplasms. However, a small percentage of lymphomas, particularly some large cell or immunoblastic B-cell tumors, will not label with LCA antibody. Other antibodies, directed against B lymphocytes, experience problems of specificity and a lack of sensitivity when applied to formalin-fixed specimens. The authors recently investigated a monoclonal antibody (L26) that demonstrates excellent specificity and sensitivity for B lymphocytes, and tumors derived from them, in formalin- and B5-fixed, paraffin-embedded tissue. The avidin-biotin peroxidase complex (ABC) technique was utilized for immunostaining 95 cases of malignant lymphoproliferative disorders and a variety of normal and neoplastic nonlymphoid tissues. When applied to sections of benign lymphoid tissue, the L26 antibody labeled germinal center cells, mantle zone and scattered interfollicular lymphocytes, but not histiocytes or plasma cells. L26 marked 100% (44/44) of the large cell and immunoblastic B-cell lymphomas, along with 1 case of pre-B cell lymphoblastic lymphoma. This included 8 cases that were LCA-negative. None of the T-cell lymphomas or plasma cell tumors studied demonstrated L26 immunostaining. No normal, benign, or neoplastic nonlymphoid tissues examined stained with this antibody. L26 successfully labels B lymphocytes and B-cell lymphomas in routinely processed tissues, often with greater sensitivity and intensity than LCA. This antibody should prove invaluable in the investigation of atypical lymphoid proliferations and the identification of B-cell derived lymphomas, when fresh or frozen tissue is unavailable for analysis.

Antibodies, Monoclonal↗

PubMed1994

Immunohistochemical detection of estrogen and progesterone receptors in endometriotic tissue. A comparative study of paraffin embedded and fresh frozen tissues.

30 formalin-fixed and paraffin embedded and 20 fresh frozen samples of endometriotic tissue were analysed immunohistochemically for the concentration of estrogen and progesterone receptors. In the formalin-fixed and paraffin embedded group only 37% of the samples were estrogen receptor positive whereas 63% were receptor negative. In contrast, we found that 67% of the samples had a positive progesterone receptor status. In the fresh frozen group 60% of endometriotic tissues were estrogen receptor positive and 75% of the tissues had a positive progesterone receptor status. We could not find any correlation between the site or severity of the endometriosis or the hormonal receptor status. We were able to demonstrate that the immunohistochemical detection of hormonal receptors in endometriotic tissues is possible and that better results were obtained if fresh frozen rather than formalin-fixed and paraffin embedded tissues were analyzed.

Endometriosis↗

PubMed2002

Detection of angiotensin-converting enzyme gene insertion/deletion polymorphism from paraffin-embedded tissues: the Hisayama study.

Previous studies have suggested that archival materials from formalin-fixed paraffin-embedded blocks are unsuitable for most molecular techniques because the extracted DNA can be severely degraded. Therefore, the present study was designed to investigate the accuracy of genotyping for the insertion (I)/deletion (D) polymorphism of the angiotensin-converting enzyme (ACE) gene from paraffin-embedded tissues of autopsy cases from Hisayama Town, Japan. The genotype was determined using the double polymerase chain reaction method and to test the accuracy of the method, the polymorphism was investigated using paraffin-embedded tissues from 18 cases whose ACE genotypes (6 cases for each genotype) were known in advance from analysis of fresh-frozen tissue samples. Genotyping using paraffin-embedded tissues was then determined for 968 autopsy subjects. The genotype could be determined in 16 of the 18 test samples (88.9%) and there was no discrepancy with the results obtained from the fresh-frozen tissues. Of the 968 autopsy cases, the frequency of the DD, ID, and II genotypes was 12.4%, 47.3%, and 40.3%, respectively, a distribution that did not deviate from the Hardy-Weinberg equilibrium (chi(22df )= 0.67, p=0.72). These findings suggest the accuracy of the present method of ACE genotyping from paraffin-embedded tissues.

Autopsy↗

PubMedDate not supplied

[Usefulness of paraffin embedding as complementary technic in the diagnosis of thyroid aspirates].

PURPOSE: To compare the diagnostic value of conventional direct cytologic smears and paraffin embedding in the preparation of samples obtained by fine needle aspiration (FNA) biopsy. METHODS: Direct smears and paraffin embedding preparations were examined from 100 thyroid lesions. In 9 cases, the FNA smears and paraffin embedding blocks were, in addition, compared with surgical thyroid specimens. RESULTS: The FNA samples were adequate for diagnosis in the great majority of cases (93/100). In 72 aspirates, both smears and paraffin embedding preparations were adequate. In 17 cases only he latter preparations and in 4 specimens only the smears were considered adequate. When both smears and paraffin blocks were available (72 aspirates), there was diagnostic agreement in 63 cases. Overall the diagnostic percentage of FNA/paraffin-block preparations was 95.7% and of FNA/conventional smears was 81.7%. Statistical analysis demonstrated that paraffin blocks increased the diagnostic accuracy of thyroid FNA (p < 0.01). CONCLUSION: Our data point out to the value of complementing conventional FNA smears with paraffin embedding preparations for the diagnosis of thyroid lesions.

Adolescent↗

PubMed2004

High-resolution cDNA microarray CGH mapping of genomic imbalances in osteosarcoma using formalin-fixed paraffin-embedded tissue.

Formalin-fixed paraffin embedded (FFPE) tumor tissue provides an opportunity to perform retrospective genomic studies of tumors in which chromosomal imbalances are strongly associated with oncogenesis. The application of comparative genomic hybridization (CGH) has led to the rapid accumulation of cytogenetic information on osteosarcoma (OS); however, the limited resolving power of metaphase CGH does not permit precise mapping of imbalances. Array CGH allows quantitative detection and more precise delineation of copy number aberrations in tumors. Unfortunately the high cost and lower density of BACs on available commercial arrays has limited the ability to comprehensively profile copy number changes in tumors such as OS that are recurrently subject to genomic imbalance. In this study a cDNA/EST microarray including 18,980 human cDNAs (which represent all 22 pairs of autosomal chromosomes and chromosome X) was used for CGH analysis of eight OS FFPE. Chromosomes 1, 12, 17, and X harbored the most imbalances. Gain/amplification of X was observed in 4/8 OS, and in keeping with other recent genomic analyses of OS, gain/amplification of 17p11.2 was often accompanied by a distal deletion in the region of the p53 gene. Gain/amplification of the X chromosome was verified using interphase FISH carried out on a subset of OS FFPE sections and OS tissue arrays.

Adolescent↗

PubMed1997

The effect of fixation type on DNA extracted from paraffin-embedded tissue for PCR studies in dermatopathology.

BACKGROUND: Amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction (PCR) is widely used to detect viral genomes, clonal gene rearrangements and oncogene mutations in skin specimens. Fixation with embedding of skin tissue is a procedure that has a profound effect on its molecular arrangement. OBJECTIVE: The aim of this study was to determine the effect of different fixatives on the PCR amplification of DNA. METHODS: We fixed randomly chosen fresh pathologic skin specimens in formalin, ethanol and Histochoice for 24 and 72 h and then embedded the tissue in paraffin. DNA was extracted from the paraffin-embedded tissues and used as template for amplification, producing 530- and 760-bp fragments of the phosphoglycerokinase gene. RESULTS: Our results indicate that PCR can be performed with excellent results on ethanol- and Histochoice-fixed, paraffin-embedded skin tissue with a rate of success comparable to that using fresh tissues; formalin-fixed tissue gave slightly less satisfactory results. CONCLUSION: This investigation corroborates previous reports investigating the effect of ethanol and formalin fixation on DNA amplification by PCR. Moreover, this is the first study showing that DNA extracted from tissue fixed with Histochoice is suitable for PCR gene amplification.

Base Sequence↗

PubMed2000

Quality control and sensitivity of polymerase chain reaction techniques for the assessment of immunoglobulin heavy chain gene rearrangements from fixed- and paraffin-embedded samples.

Frozen tissue is considered the gold standard if DNA is to be extracted for polymerase chain reaction (PCR) analysis. In molecular studies from paraffin-embedded material, only positive results are usually taken into account. Our goal was to evaluate both the sensitivity and the specificity of PCR techniques for immunoglobulin heavy chain (IgH) gene rearrangement according to various lengths and types of fixative before paraffin embedding. One set of studies compared IgH rearrangement in a case of mantle cell lymphoma tissue that had been fixed in 14 different ways before paraffin embedding and frozen tissue. Formalin fixation was found not to be deleterious for DNA, amplification being possible up to 15 days after fixation with good sensitivity. In contrast, the performance of PCR decreased for samples fixed in Bouin's liquid for longer than 6 hours or after 48 hours of incubation in a vacuum infiltration processor (in which Bouin's liquid-fixed and formalin-fixed samples are mixed). In addition, we undertook a retrospective study of 20 routinely processed B-cell lymphomas, with frozen formalin-fixed and Bouin's liquid-fixed tissues for each case. Of the 14 positive cases on frozen material, 13 were also clonal from paraffin-embedded tissues. Whatever the IgH locus analyzed, each time the adapted control was positive, results from paraffin-embedded material were identical to results obtained from frozen tissue. In this study, we showed that the use of paraffin-embedded tissue is efficient for the study of IgH gene rearrangement. Whenever adapted controls are used, it is even possible to assess negative results.

DNA Primers↗

PubMed1997

[DNA flow cytometry. Comparison of the methods for nuclear extraction from frozen and paraffin-embedded sections].

Cellular DNA content of solid tumors can be determined either from fresh, frozen, or formalin fixed, paraffin-embedded tissues. However, discordant results have been obtained using the paraffin-embedded technique, and lack of abnormal DNA stemlines in the paraffin-embedded as compared to either fresh or frozen tissues has been reported. In this study we evaluated the validity of nuclear extraction method from paraffin-embedded tissues, using 75 breast carcinomas whose DNA content was previously analyzed from frozen tissue and resulted either normal (12 cases) or abnormal (63 cases). From representative paraffin blocks, nuclei were extracted following Hedley's technique. The results revealed excellent cell counting and good histogram resolution from all paraffin samples; the loss of G2M abnormal peak in eight histograms with abnormal stemline did not compromise the correct interpretation of DNA content. In addition, the comparison between DNA indices obtained from corresponding paraffin and frozen samples showed a good correlation in 69 cases (r = 94); discordance in six cases was demonstrated to be related to tumor heterogeneity. In conclusion the paraffin extraction method is a sensible, and reliable technique, which can be applied for DNA flow cytometric studies on archival cases, as well as whenever fresh sample from the tumor is not obtainable.

Aneuploidy↗

PubMed2000

PCR-heteroduplex analysis of T-cell receptor gamma gene rearrangement in paraffin-embedded skin biopsies.

We developed a rapid, simple, and sensitive method for the detection of T-cell receptor-gamma (TCRgamma) gene rearrangements in paraffin-embedded skin biopsies. Available techniques often require either fresh tissue, several primer pairs, nested amplifications, or specialized electrophoresis steps such as denaturing gradient gel electrophoresis. Our method is based on heteroduplex analysis of polymerase chain reaction (PCR) products of the TCRgamma in a nondenaturing modified polyacrylamide gel using a single pair of primers and is adapted for paraffin-embedded tissue. When tested against Southern blot analysis, the PCR results correlated in 8 of 9 cases. Six mature cutaneous B-cell lymphomas and 29 inflammatory skin disorders all resulted in a polyclonal amplification pattern. When analyzing 3-mm or 4-mm punch biopsies of 51 cases of cutaneous T-cell lymphoma, 37 (72.5%) showed a clonal rearrangement with this technique. For 7 cases of patch stage mycosis fungoides, frozen tissue and formalin-fixed and paraffin-embedded tissue was available, and in 5 of 7 cases (71%), the results in frozen and paraffin-embedded tissue were concordant. One case showed a clonal pattern in frozen tissue but not in paraffin-embedded tissue, and one case was polyclonal in frozen tissue but monoclonal in paraffin-embedded tissue. Using serial dilutions of DNA from a T-cell ALL in a polyclonal background (tonsil), we established a sensitivity of 0.5%. Heteroduplex PCR of the TCRgamma is a rapid, sensitive, and inexpensive screening procedure as well as a useful adjunct to histologic analysis and immunophenotyping of cutaneous T-cell proliferations.

Biopsy, Needle↗

PubMed1992

Variation in reference cells for DNA analysis of paraffin-embedded tissue.

Selection of the diploid reference cells used in flow cytometric DNA analysis of paraffin-embedded tissue is inconsistent in the literature. To determine which types of cells were most suitable for use as reference cells, benign paraffin-embedded tissue was evaluated from nine randomly selected autopsies. Benign kidney, lymph node, gastrointestinal mucosa, laryngeal mucosa, bronchial mucosa, bladder mucosa, pancreas, and, when available, prostate tissue were studied. Ten paraffin-embedded surgical specimens also were studied. In the autopsy specimens, great variability in the mean peak channels was noted on intrapatient evaluation and even more variability was present when comparing similar organs (especially lymph nodes) from different patients. Similar results were obtained using lymph nodes from surgical specimens. It is concluded that the most suitable diploid reference cells for DNA analysis of paraffin-embedded tissue are the benign cells present in the paraffin tumor block that have been processed in the same way as the tumor cells.

Aged↗

PubMed1999

Heat pretreatment increases resolution in DNA flow cytometry of paraffin-embedded tumor tissue.

BACKGROUND: Flow cytometry of single-cell suspensions prepared by enzymatic digestion from formalin-fixed, paraffin-embedded tissue suffers from several major drawbacks. The most important factors that influence the results are the high and unpredictable coefficients of variation (CVs) of the G0/G1 peak in the DNA histogram and reduction of propidium iodide (PI) intercalation with DNA, resulting from protein cross-linking by formalin. METHODS: In this study we introduce a heating step (2 h incubation in citrate solution at 80 degrees C) prior to a brief pepsin digestion of tissue sections in the protocol for DNA content analysis of formalin-fixed and paraffin-embedded tissue. This new method is compared with established methods for the preparation of cell suspensions from frozen and paraffin-embedded tissues with respect to cell yield, DNA histogram resolution, DNA dye saturation kinetics, cell cycle parameters, and antigen retrieval in various epithelial and nonepithelial tissues. RESULTS: The recovery of single cells from the paraffin sections was doubled by the heat treatment step, while the limited time of proteolysis resulted in decreased cell debris. Furthermore, an increased fraction of cells became cytokeratin-positive, while these immunocytochemically stained cells also exhibited a higher mean fluorescence intensity. The DNA histograms prepared from cell suspensions obtained according to this new protocol showed a significantly improved resolution, leading to a better identification of peridiploid cell populations. Heat pretreatment of paraffin-embedded archival tissue sections showed PI saturation kinetics similar to, or even better than, those of fresh unfixed tissues, independent of duration of fixation. CONCLUSIONS: This new method, making use of routinely available antigen retrieval principles, thus allows high-resolution DNA analysis of routinely fixed and paraffin-embedded tissue samples. Using external reference cells, inter- and intralaboratory standardization of DNA histograms can be achieved.

Breast Neoplasms↗

PubMed1994

Effects of fixative and fixation time on the extraction and polymerase chain reaction amplification of RNA from paraffin-embedded tissue. Comparison of two housekeeping gene mRNA controls.

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.

Animals↗

PubMed1998

Mutations in BRCA1 from fixed, paraffin-embedded tissue can be artifacts of preservation.

DNA isolated from paraffin-embedded tissues has been used for analysis of DNA alterations in disease states. Use of archival tissue can expedite the gathering of large numbers of specimens from rare disease subtypes that would take years to accumulate prospectively. Therefore, archival tissues from 70 ovarian cancer cases diagnosed before or at age 40 were retrieved for analysis of BRCA1 mutations. DNA was isolated from paraffin-embedded tissue of 70 ovarian cancer cases diagnosed before or at age 40. BRCA1 mutation analysis was conducted by single-strand conformation polymorphism analysis and DNA sequencing. Fifty-eight BRCA1 mutations were found in 34 of the 70 ovarian cancer cases. Twenty-two cases had one mutation each and 12 cases had multiple mutations. Multiple mutations found in histologically normal tissue of 2 cases were not present in matched tumor tissue. For another case, DNA from two separate blocks of normal tissue contained different mutations. These observations were anomalous and suggested that mutations detected in fixed tissues may be artifacts of tissue preservation and not present in the original unfixed tissues. To test this suggestion, blood was obtained from 2 patients for whom mutations were found in fixed, normal tissue. DNA from their unfixed lymphocytes did not contain the mutations found in fixed normal tissue. Thus, mutations found in fixed, paraffin-embedded tissues can be artifacts of tissue preservation. The reliability of DNA sequence data derived from such tissues must be questioned in the absence of corroborating data from unfixed tissues. This severely limits the use of fixed tissues as a source of DNA for retrospective research and for clinical genetic testing in families for which a disease-affected member is not alive.

Adult↗