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PubMed1994

Extraction of DNA from paraffin-embedded tissue for analysis by polymerase chain reaction: new tricks from an old friend.

For decades virtually all human tissues have been fixed in formalin and embedded in paraffin. The DNA present in fixed tissues is degraded in size but can be analyzed by the polymerase chain reaction (PCR) for specific genetic alterations. The original purpose of fixation, to optimally preserve complex histological and spatial features, is lost with bulk DNA extraction. Because the PCR allows the analysis of even a single molecule of DNA, it is possible to genetically analyze small groups of cells on a microscope slide with techniques such as selective ultraviolet radiation fractionation (SURF). The direct correlation of histological phenotype with genotype provides information that is difficult to obtain by other methods and allows a more precise translation between molecular studies and the current vast histological database.

Chemical Fractionation↗
PubMed2000

Molecular cytogenetic studies in three patients with partial trisomy 2p, including CGH from paraffin-embedded tissue.

We report on three cases of partial trisomy 2p in which the identification and exact localization of the duplicated chromosome segment was possible only by application of molecular cytogenetic techniques. These included fluorescence in situ hybridization by use of wcp2, N-myc, and subtelomeric 2p probes and comparative genomic hybridization with DNA isolated from blood samples, frozen fetal tendon, and formalin fixed, paraffin-embedded fetal lung tissue. Two of the cases concerned fetuses of gestational week 20 and 24 with duplication of nonoverlapping terminal (2pter-->p24) and more proximal (2p25-->p23) segments and with distinctly different phenotypes. The third case was due to a de novo inverted duplication of 2p25-->p23, with loss of the subtelomeric region of 2p. This 53-month-old girl was a Bloom syndrome carrier. The patient had prenatal growth failure, borderline microcephaly, dilated lateral horns of the cerebral ventricles, transient cortical blindness, myopia, muscle hypotonia, and dilatation of the left renal collecting system. Dermal cysts were found on the glabella, the soles of both feet, and the vocal cord, causing respiratory embarrassment. Previously reported cases of pure trisomy 2p are reviewed, in an attempt to correlate clinical findings to overlapping regions in 2p. These cases illustrate the effectiveness of molecular cytogenetic methods in resolving subtle chromosomal aberrations in order to coordinate more accurately a chromosome regionspecific phenotype.

Abortion, Induced↗
PubMed1997

Numerical aberrations of chromosome 7 detected in 15 microns paraffin-embedded tissue sections of primary cutaneous melanomas by fluorescence in situ hybridization and confocal laser scanning microscopy.

Fluorescence in situ hybridization (FISH) to sections of formalin-fixed paraffin-embedded archival tissues allows the detection of gene or chromosome copy number changes in interphase cell nuclei within the histological context and thus may be of particular interest in tumor pathology. In this report, we describe the application of FISH to thick (15 microns) paraffin sections of 7 primary cutaneous malignant melanomas. A chromosome 7-specific centromeric DNA probe was used to detect numerical aberrations of chromosome 7. By optical sectioning using confocal laser scanning microscopy (CLSM) only complete, uncut interphase cell nuclei were scored. The mean percentage (+/- SEM) of melanoma cell nuclei with three hybridization spots was (20.7 +/- 2.8)%; (6.8 +/- 1.0)% of nuclei showed one spot and (5.0 +/- 1.2)% four or more spots. The frequency distribution of spot numbers among melanoma cell nuclei and normal keratinocyte nuclei was significantly different (chi(2) = 176.8, df = 5, p < 0.001). Trisomy 7 was detected in all 7 cases analyzed, mostly associated with monosomy 7 or polysomy 7. The approach used in our study and the data obtained could be useful for further studies designed to investigate a possible involvement of chromosome 7 in melanocytic tumor progression.

Adult↗
PubMedDate not supplied

Flow-cytometric analysis of the DNA content in paraffin-embedded tissue and in fresh tumour samples obtained from renal cell carcinoma: comparison of DNA ploidy using ethidium bromide and mithramycin.

With the help of the deparaffination technique described by Heldly, it has become possible to conduct large-scale flow-cytometric (FCM) studies, including retrospective DNA analysis of renal cell carcinoma (RCC). The quality of these histograms, however, has to be comparable to the quality of the analyses from fresh tumour tissue. The present study compares the application of FCM on fresh tumour tissue from RCC with paraffin-embedded archive material, thus for the first time, the same combination of stains (ethidium bromide/mithramycin) was used for both materials. From a total of 44 tumours, 125 histograms were obtained from fresh tumour tissue. Aneuploidy was found in 78 (62.4%) of the DNA analyses (coefficient of variation, CV = 5.5%). These 78 aneuploid histograms were compared with 228 sections from the archive material which had been deparaffined and stained. This involved, a deparaffinising period of twelve hours as well the application of pepsin and protease, which was necessary to obtain high-quality histograms (CV = 6.0%). Retrospectively, a total of 58 histograms from 36 cases were available for comparison. Of the archive material, 23/36 tumours were classified as aneuploid; thus new stem cell lines could be detected retrospectively in 63.8% of the cases. The comparison of DNA indices (DI) in the two series produced no significant differences, and the comparison of the mean values for the CV-5.5% for native material histograms and 6.0% for archive material histograms-was not statistically significant. The detritus part in the archive histograms revealed a mean value of 6.3% for native histograms the mean value was 13.4%. These differences were also not significant. These results show that, using the staining combination ethidium bromide/mithramycin, the quality of the histograms obtained with archive material is equal to the quality of the histograms obtained from fresh tumour material. Furthermore, when taking into consideration the clonal heterogeneity of RCC, it is possible to use retrospective analysis to detect aneuploidy with a high degree of certainty and accuracy.

Carcinoma, Renal Cell↗
PubMed1992

Detection of c-erbB-2 activation in paraffin-embedded tissue by immunohistochemistry.

Commercially available monoclonal antibodies were tested for their ability to detect increased levels of c-erbB-2 protein in formalin-fixed, paraffin-embedded breast carcinomas. Of five antibodies studied, four (TAB-250, CB11, 3B5, and N3/D10) showed strong cytoplasmic membrane reactivity in 23% (11 of 47) of routinely processed tumors, although interpretation of the immunoreactivity with 3B5 and N3/D10 occasionally was difficult due to cytoplasmic granular staining. Since the c-erbB-2 oncogene is activated by DNA amplification and overexpression of mRNA and protein, the same tumors were analyzed for c-erbB-2 activation by other techniques. c-erbB-2 activation in these 11 tumors was confirmed by immunohistochemistry of frozen tissue (nine of nine tumors), in situ hybridization (nine of 11 tumors), and Southern blot analysis (five of eight tumors). In some of these tumors the failure to demonstrate c-erbB-2 DNA amplification may be due to the small percentage of malignant cells. One additional tumor showed probable c-erbB-2 protein overproduction based on strong immunoreactivity with two antibodies (TAB-250 and CB11), although no definite activation could be demonstrated by additional techniques. Three other tumors (6%) showed equivocal c-erbB-2 protein overproduction based on weak immunoreactivity only with TAB-250, although unequivocal activation could not be demonstrated by additional techniques. The 32 carcinomas (68%) that showed no significant immunoreactivity with any antibodies in routinely processed tissue also showed no detectable c-erbB-2 activation by additional techniques. We conclude that TAB-250 and CB11 are reliable antibodies for detecting c-erbB-2 protein overproduction in routinely processed tissue. TAB-250 also weakly stains a few tumors showing no definite c-erbB-2 activation by other techniques. Two additional antibodies (3B5 and N3/D10) detect c-erbB-2 protein overproduction in paraffin-embedded tissue, but are more difficult to interpret. A fifth antibody, TA-1, is an excellent reagent for use on frozen tissue, but prolonged formalin fixation may impair recognition of its antigenic epitope.

Adenocarcinoma↗
PubMed1994

Adenovirus myocarditis: retrospective diagnosis by gene amplification from formalin-fixed, paraffin-embedded tissues.

Understanding the pathogenesis of viral myocarditis is linked to the availability of sensitive assays to detect viruses in clinical material. Recent advances in molecular techniques permit direct detection of viral-specific nucleic acid in tissue samples. This report describes a protocol for DNA extraction and amplification of adenovirus genome from formalin-fixed, paraffin-embedded human tissues that detects as little as 10 copies of viral genome in a background of 0.5 micrograms of human DNA. This sensitive assay permitted the examination of archived tissues to establish a retrospective diagnosis of adenoviral myocarditis in two pediatric patients.

Adenovirus Infections, Human↗
PubMed2004

Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay.

Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10- micro m FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.

Artifacts↗
PubMed2006

Unravelling the proteome of formalin-fixed paraffin-embedded tissue.

Biofluid detectable biomarkers that originate at the site of diseased tissues would be advantageous, in that, they may provide mechanistic information concerning the manifestation and progression of the disease. Unfortunately, tissue biopsies are precious samples that can generally be acquired in small amounts due to the invasive nature of the sample collection. One of the foundations of pathological diagnosis for decades has been from formalin-fixed paraffin-embedded (FFPE) tissues, of which a vast archive exists worldwide. These tissues have also been widely used for immunohistochemistry and in situ hybridization studies examining for expression of specific proteins or transcripts. Unfortunately, the ability to analyse FFPE tissues using mass spectrometry (MS) has been essentially non-existent until recently. In this review, methods that allow the extraction of peptides from FFPE tissues and their proteomic analysis using MS are described. The ability to identify the proteins extracted from FFPE tissues allows comparative analyses that enable the potential discovery of novel biomarkers at the site of the diseased tissue.

Animals↗
PubMed2006

Application of array CGH on archival formalin-fixed paraffin-embedded tissues including small numbers of microdissected cells.

Array-based comparative genomic hybridisation (aCGH) has diverse applications in cancer gene discovery and translational research. Currently, aCGH is performed primarily using high molecular weight DNA samples and its application to formalin-fixed and paraffin-embedded (FFPE) tissues remains to be established. To explore how aCGH can be reliably applied to archival FFPE tissues and whether it is possible to apply aCGH to small numbers of cells microdissected from FFPE tissue sections, we have systematically performed aCGH on 15 pairs of matched frozen and FFPE astrocytic tumour tissues using a well-established in-house human 1 Mb BAC/PAC genomic array. By spiking tumour DNA with normal DNA, we demonstrated that at least 70% of tumour DNA was required for reliable aCGH analysis. Using aCGH data from frozen tissue as a reference, it was found that only FFPE astrocytic tumour tissues that supported PCR amplification of >300 bp DNA fragment provided high quality, reproducible aCGH data. The presence of necrosis in a tissue specimen had an adverse effect on the quality of aCGH, while fixation in formalin for up to 96 h of fresh tissue did not appear to affect the quality of the result. As little as 10-20 ng DNA from frozen or FFPE tissues could be readily used for aCGH analysis following whole genome amplification (WGA). Furthermore, as few as 2000 microdissected cells from haematoxylin-stained slides of archival FFPE tissues could be successfully used for aCGH investigations when WGA was used. By careful assessment of DNA integrity and review of histology, to exclude necrosis and select specimens with a high proportion of tumour cells, it is feasible to preselect archival FFPE tissues adequate for aCGH analysis. With the help of microdissection and WGA, it is also possible to apply aCGH to histologically defined lesions, such as carcinoma in situ.

Astrocytoma↗
PubMed2004

The HOPE-technique permits Northern blot and microarray analyses in paraffin-embedded tissues.

There is an increasing demand for tissue samples that, after having been used for conventional histologic examination, are also suited for molecular analyses. As to formalin-fixed, paraffin embedded (FFPE) tissue, the latter applications are very limited. The HOPE (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) technique comprises a new protection-solution with an organic buffer, with acetone as the only dehydrating agent, and pure paraffin of 52-54 degrees C melting temperature, allowing for all pathologic routine investigations. In contrast to FFPE tissue, the HOPE-technique allows for the application of molecular methods, such as high molecular DNA and RNA isolation, which can be used for PCR and reverse transcription PCR (RT-PCR). In this study, we investigated whether RNA from HOPE-fixed tissue samples is suitable for Northern blot and microarray analyses. RNAs of two HOPE-fixed breast cancer specimens of different histologic grade were used to carry out an array experiment. It turned out that RNA from HOPE-fixed tissue is of high quality and can be successfully used for array experiments. In addition, by detecting GAPDH and high mobility group protein gene B1 (HMGB1)-specific transcripts, we were able to demonstrate that RNA from HOPE-fixed tissue can also be used for Northern blot hybridization.

Blotting, Northern↗
PubMed2006

Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry.

A satisfactory protocol of protein extraction has been established based on the heat-induced antigen retrieval (AR) technique widely applied in immunohistochemistry for archival formalin-fixed, paraffin-embedded (FFPE) tissue sections. Based on AR, an initial serial experiment to identify an optimal protocol of heat-induced protein extraction was carried out using FFPE mouse tissues. The optimal protocol for extraction of proteins was then performed on an archival FFPE tissue of human renal carcinoma. FFPE sections were boiled in a retrieval solution of Tris-HCl containing 2% SDS, followed by incubation. Fresh tissue taken from the same case of renal carcinoma was processed for extraction of proteins by a conventional method using radioimmunoprecipitation assay solution, to compare the efficiency of protein extraction from FFPE tissue sections with extraction from fresh tissue. As a control, further sections of the same FFPE sample were processed by the same procedure without heating treatment. Evaluation of the quality of protein extracted from FFPE tissue was done using gel electrophoresis and mass spectrometry, showing most identified proteins extracted from FFPE tissue sections were overlapped with those extracted from fresh tissue.

Animals↗
PubMed1996

[Immunohistochemical labeling of inner ear tissues embedded in polyethylene glycol 4000--comparative study with araldite and unicryl embedded sections].

An improved method for embedding specimens in polyethylene glycol (PEG) 4000, a water soluble polymer, was used to prepare the vestibular end-organs of the inner ear. Staining of the tissue sections of PEG embedded specimens with antibodies to alpha-tubulin and to calmodulin was compared with staining of tissue sections of Araldite and Unicryl embedded specimens. PEG embedded sections revealed sensitive immunocytochemical labeling with excellent morphological resolution. The problems of embedding and orienting small specimens of the inner ear in PEG are described and the methods used to solve them are described.

Acrylic Resins↗
PubMed1984

Histone RNA in amphibian oocytes visualized by in situ hybridization to methacrylate-embedded tissue sections.

We present an in situ hybridization method for detecting cellular RNAs in tissue sections using methacrylate as the embedding medium. The technique offers the advantage of superior morphological preservation compared with previously published procedures. Since sections can be cut 1 micron or less in thickness, full advantage is taken of the short path length of 3H electrons. Applying this procedure to developing amphibian oocytes, we investigated the accumulation and localization of RNA complementary to the histone genes and their adjacent spacers. Histone RNA begins to accumulate in the cytoplasm of late pachytene-early diplotene oocytes, rapidly reaching a maximum concentration during Dumont stage 1. After this stage the concentration of histone RNA declines. RNA transcribed from histone coding regions is located almost exclusively in the cytoplasm of oocytes. Transcripts of the spacer regions, which are known to be synthesized on oocyte lampbrush chromosomes, do not accumulate in the oocytes. [3H]RNA complementary to U2 small nuclear RNA, used in these experiments as a control, hybridized predominantly to the nucleus of the oocytes.

Animals↗
PubMed2005

Primary local orbital amyloidosis: biochemical identification of the immunoglobulin light chain kappaIII subtype in a small formalin fixed, paraffin wax embedded tissue sample.

BACKGROUND: Amyloidosis refers to a heterogeneous group of disorders associated with the deposition of chemically distinct amyloid fibril proteins. Precise determination of chemical amyloid type has diagnostic, therapeutic, and prognostic relevance. Although immunohistochemical techniques are used routinely to determine the amyloid type, the results can be negative or inconclusive, so that biochemical characterisation is often required. The development and application of new biochemical microtechniques suitable for examination of extremely small tissue samples is essential for precise identification of the deposited amyloid proteins. AIMS: To investigate biochemically the amyloid proteins present in a formalin fixed paraffin wax embedded orbital tissue from a patient with localised orbital amyloidosis in whom immunohistochemistry was not helpful in the determination of amyloid type. METHODS: Extraction of amyloid proteins from fixed tissue and their identification was carried out by a recently developed microtechnique. An extremely small tissue sample was dewaxed and extracted with formic acid. The extracted material was analysed using electrophoresis, western blotting, and amino acid sequencing. RESULTS: Biochemical examination of the extracted proteins showed the presence of immunoglobulin (Ig) derived amyloid proteins, which were composed of the N-terminal fragments of the Ig light chain kappaIII subtype (AL-kappaIII) (16, 8, and 3 kDa). CONCLUSIONS: This is the first chemically proved AL case reported in association with primary localised orbital amyloidosis. The biochemical microtechnique used was useful in achieving a precise diagnosis of amyloid disease, in a case where the results of routine immunohistochemical examination of amyloid were inconclusive.

Adult↗
PubMed2000

Myogenin is a specific marker for rhabdomyosarcoma: an immunohistochemical study in paraffin-embedded tissues.

Myogenin belongs to a group of myogenic regulatory proteins whose expression determines commitment and differentiation of primitive mesenchymal cells into skeletal muscle. The expression of myogenin has been demonstrated to be extremely specific for rhabdomyoblastic differentiation, which makes it a useful marker in the differential diagnosis of rhabdomyosarcomas (RMS) from other malignant small round cell tumors of childhood. Commercially available antibodies capable of detecting myogenin in routinely processed formalin-fixed paraffin-embedded (FFPE) tissue are now available. In this study, we evaluated myogenin expression using the monoclonal myf-4 antibody (Novocastra Labs) on FFPE in a large number of pediatric tumors in order to define the clinical utility of this marker. A total of 119 tumors were studied. These included 48 alveolar RMS (ARMS), 20 embryonal RMS (ERMS), one spindle cell RMS, 16 Ewing's sarcomas (ES), six nephroblastomas, two ectomesenchymomas, seven precursor hematopoietic neoplasms, five olfactory neuroblastomas, three neuroblastomas, six desmoplastic small round cell tumors, and five rhabdoid tumors. Distinct nuclear staining for myogenin was noted in all 69 RMS. Notably, the number of positive tumor cells differed between the ARMS and ERMS. In ARMS, the majority of tumor cells (75 to 100%) were positive, in contrast to ERMS, in which the positivity ranged from rare + to 25% in all but three tumors. Additionally, myogenin positivity was seen in two of two ectomesenchymomas and in two nephroblastomas with myogenous differentiation. All other tumors were clearly negative. Our results indicate that staining for myogenin is an extremely reliable and specific marker for rhabdomyoblastic differentiation. It gives consistent and easily interpretable results in routinely fixed tissues.

Biomarkers, Tumor↗
PubMed1976

Sex chromatin determination in formalin-fixed, paraffin-embedded tissues of rats.

Distinctive chromatin bodies appearing in interphase nuclei of mammalian female cells have been found in most mammalian species. The positive correlation between sex chromatin masses and mammalian female tissue presents an almost ideal criterion for sex determination of mammalian tissues. Previous studies indicate that this positive correlation exists in fresh and formalin-fixed, paraffin-embedded normal and tumor tissue of humans, as well as in fresh squash preparations of normal and tumor tissues of rodents. In this experiment we have shown that it is also possible to determine sex in formalin-fixed, paraffin-embedded normal and tumor tissue of the rat.

Adenocarcinoma↗