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PubMed1993

Detection of clonal B cell populations in paraffin-embedded tissues by polymerase chain reaction.

A method was established to detect clonal B cell populations in frozen and paraffin-embedded tissues. The method is based on the polymerase chain reaction amplification of rearranged VH and V kappa genes using V gene family-specific primers. Monoclonal B cell populations could be detected in peripheral blood lymphocyte DNA in all 16 cases of B-CLL and immunocytoma investigated and in 8 of 10 cases of B cell non-Hodgkin's lymphoma using frozen and paraffin sections. The amplification of V kappa rearrangements in addition to the VH amplification is a useful tool to verify the results of the heavy chain rearrangement and to detect proliferation of a B cell clone in cases in which no VH product was obtained. In spite of the degradation of DNA in paraffin-embedded tissues, we were able to find amplified polymerase chain reaction products of about 350 bp length in 8 of 10 cases analyzed. The method presented here may be helpful in routine diagnosis of B cell non-Hodgkin's lymphoma using frozen or paraffin-embedded specimens.

B-Lymphocyte Subsets↗
PubMed2001

Congenital-infantile fibrosarcoma. A clinicopathologic study of 10 cases and molecular detection of the ETV6-NTRK3 fusion transcripts using paraffin-embedded tissues.

Congenital-infantile fibrosarcoma (CIFS) is a relatively indolent sarcoma that should be distinguished from more aggressive spindle cell sarcomas of childhood. CIFSs have been found to have a novel recurrent reciprocal translocation t(12;15)(p13;q25) resulting in the gene fusion ETV6-NTRK3 (ETS variant gene 6; neurotrophic tyrosine kinase receptor type 3). We studied immunohistochemical expression of NTRK3, and conducted a reverse transcription-polymerase chain reaction (RT-PCR) assay to detect the ETV6-NTRK3 fusion transcripts using archival formalin-fixed paraffin-embedded tissues from 10 CIFSs. Thirty-eight other spindle cell tumors were included as controls. The ETV6-NTRK3 fusion transcripts were identified in 7 (70%) of 10 CIFSs. Nucleotide sequence analysis showed that the fusion occurred between ETV6 exon 5 and NTRK3 exon 13. The 38 control tumors were negative for the fusion transcript. Immunohistochemically, CIFSs consistently expressed NTRK3. But the expression of NTRK3 also was observed in 22 of 38 control tumors. These results show the diagnostic usefulness of RT-PCR methods to detect ETV6-NTRK3 fusion transcripts in archival formalin-fixed paraffin-embedded tissue and the important role of NTRK3 in the development of CIFS, despite its being a protein of little importance in differential diagnosis.

Child↗
PubMed1988

Surface spectroscopic characterization of titanium implants after separation from plastic-embedded tissue.

The method of plastic embedding of tissue and implant and subsequent separation of plastic and implant for preparing sections of tissue adjacent to solid metallic implants relies on a successful separation of the embedment and the implant. In this work, the surface of machined Ti implants has been analysed in order to investigate to what extent plastic remnants exist on the implant after separation. SEM and AES analyses show that at least 70% of the implant surface is free of plastic remnants to a proximity of 10 nm or less from the implant surface. The method is simple and suitable for both light and transmission electron microscopy of the interface tissue.

Bone Screws↗
PubMed1989

Enzyme histochemistry on freeze-dried, resin-embedded tissue.

We have developed a method for histochemical demonstration of a wide range of enzymes in freeze-dried, resin-embedded tissue. Freeze-dried tissue specimens were embedded without fixation at low temperature (4 degrees C or -20 degrees C) in glycol methacrylate resin or LR Gold resin. Enzyme activity was optimally preserved by embedding the freeze-dried tissue in glycol methacrylate resin. All enzymes studied (oxidoreductases, esterases, peptidases, and phosphatases), except for glucose-6-phosphatase, were readily demonstrated. The enzymes displayed high activity and were accurately localized without diffusion when tissue sections were incubated in aqueous media, addition of colloid stabilizers to the incubating media not being required. Freeze-drying combined with low-temperature resin embedding permits the demonstration of a wide range of enzymes with accurate enzyme localization, high enzyme activity, and excellent tissue morphology.

Animals↗
PubMed1987

Quantitative X-ray microanalysis of calcium with the Camebax-TEM system in frozen, freeze-substituted and resin-embedded tissue sections. Application to molluscan glio-interstitial granules.

The relevance of the continuum method for a quantitative X-ray microanalysis of epon embedded tissue sections in the particular conditions offered by the Camebax-TEM system was tested and an improved model of specimen holder is proposed. The absolute calcium concentration [Ca] of membrane-bound intracellular glio-interstitial granules was determined by X-ray microanalysis in transmission electron microscopy of Mytilus retractor muscle. The Ca peak and background values were measured by the wavelength-dispersive spectrometer of the Camebax; the mass thickness of the section was recorded simultaneously with an added energy-dispersive detector. The tissue was frozen at approximately equal to 77 K in a mixture of liquid propane and butane, freeze-substituted in the presence of oxalic acid and embedded in epoxy resin. The calcium concentration of glio-interstitial granules can be as high as 180 mmol.kg-1 of epoxy-embedded tissue, with an average of 40 mmol.kg-1. The sampling of the data through repeated experiments is discussed and it is proposed that the cell would be the main level of variation. The Ca content of glio-interstitial granules is significantly lower in the tissues of animals submitted to high-potassium artificial sea-water for 10 min. This finding was predicted by the hypothesis that glio-interstitial tissue is a regulator of calcium concentration in extracellular spaces.

Animals↗
PubMed1989

A novel monoclonal antibody (OPD4) recognizing a helper/inducer T cell subset. Its application to paraffin-embedded tissues.

A novel monoclonal antibody (MAb), OPD4, reactive with a helper/inducer (H/I) subset of T cells in formalin-fixed, paraffin-embedded tissue sections, has been identified through immunization with an activated H/I T cell line, namely DL40. The antibody is an IgG1 antibody and it recognizes an antigen with a molecular weight of 200 kd, corresponding to that of leukocyte common antigen. OPD4+/CD4+ T cells provided better help for pokeweed mitogen-stimulated polyclonal IgG production than OPD4-/CD4+ T cells. OPD4 recognized the H/I T cell subset even in paraffin-embedded tissue sections, but did not recognize nonhematopoietic cells, suppressor/cytotoxic T cells, B cells, monocytes in the peripheral blood, or other normal hematopoietic cells as examined by the flow cytometric and immunoperoxidase methods. Besides the lymphoid cells, OPD4 reacted with a number of histiocytes (epithelioid cells) in tissues from sarcoidosis and tuberculosis. For the neoplastic lesions, OPD4 reacted with approximately half of the cases of T cell lymphomas. Consequently, OPD4 may be useful for the diagnosis and study of malignant lymphomas and other related lesions.

Antibodies, Monoclonal↗
PubMed2002

Comparison of techniques for the successful detection of BRCA1 mutations in fixed paraffin-embedded tissue.

Genomic DNA isolated from archived paraffin-embedded tissues (PETs) has important applicability in genetic epidemiological studies. To determine the accuracy of the sequence data, using DNA derived from PET among patients with known mutations characterized from blood, we conducted a blinded factorial experiment to simultaneously examine the influence of mutation type, age of the PET, PCR product type, and Taq DNA polymerase on BRCA1 gene mutation detection. The probability of detecting sequencing artifacts was also investigated. We found that: (a) gene detection was most accurate for newer PET; (b) high fidelity Taq with shorter PCR amplicon length yielded the highest mutation detection success rate and lowest artifact rate; and (c) base substitutions were more often correctly identified than frameshift mutations or wild-type sequences. We concluded that DNA derived from PET that archived for less than 18 years can be used successfully for detecting BRCA1 gene mutations if quality control is strictly maintained.

DNA Mutational Analysis↗
PubMed2000

The use of fluorescence in situ hybridization (FISH) on paraffin-embedded tissue sections for the study of microchimerism.

We describe here a simple and versatile method of fluorescence in situ hybridization (FISH) on paraffin-embedded tissue sections with specific application in the study of microchimerism, that is, the presence of intact foreign cells within an individual. This is accomplished through the use of X and Y chromosome-specific probes to identify the presence of male nuclei within a tissue section from a female, and vice versa. This technique requires only minor modification if at first the hybridization does not yield fluorescent signals of high quality. Analysis of a wide variety of tissue types is possible with this method, and multiple tissue types from one or more individuals can be processed in the same hybridization reaction. This robust FISH method has been used successfully in our laboratory to investigate fetal cell microchimerism in the following paraffin-embedded tissue types: skin, lung, thyroid, adrenal gland, lymph node, heart, spleen, liver, pancreas, kidney, and intestine.

Cell Nucleus↗
PubMed1989

Flow cytometry using paraffin-embedded tissue: five years on.

The use of paraffin-embedded tissue for flow cytometry is reviewed. A number of technical modifications of the original 1983 method have been described, aimed at improving the accuracy of DNA measurements by minimizing cell debris or reducing coefficients of variation, and at simplifying sample preparation. Over 100 clinical studies have now been reported, mainly assessing the effect of DNA index on prognosis, and those published up until mid-1988 are summarized in an appendix. More recently there have been developments in the use of monoclonal antibodies to measure oncogene products or proliferation markers in addition to DNA content. Detailed clinical evaluation and standardization of these more sophisticated methods is still some way ahead, but as was the case with DNA index, the use of archival material from patients whose outcome is already known should speed this process.

Animals↗
PubMed1998

A new Mel-CAM (CD146)-specific monoclonal antibody, MN-4, on paraffin-embedded tissue.

Mel-CAM (also termed CD146 or MUC18) is a cell-adhesion molecule belonging to the immunoglobulin gene superfamily. It mediates cell-cell interaction through heterophilic Mel-CAM/ligand adhesion. Previous studies showed Mel-CAM immunoreactivity in normal and neoplastic human tissues, but an extensive assessment of Mel-CAM distribution was not performed because of the lack of a Mel-CAM-specific monoclonal antibody that could be used in routinely processed, formalin-fixed, paraffin-embedded tissues. Therefore, we developed a mouse monoclonal antibody, MN-4, that specifically recognized a fixation-resistant epitope in the second extracellular (C2) domain of Mel-CAM. We performed immunohistochemical staining on 467 paraffin-embedded tissue samples with this MN-4 antibody and avidin-biotin peroxidase. MN-4 immunoreactivity was seen in normal tissues, including endothelium, smooth muscle, epithelial and myoepithelial cells of the breast, Schwann cells, ganglion cells, cerebellar cortex, implantation-site intermediate trophoblast, external root sheath of hair follicles, basal cells of bronchial epithelium, parathyroid glands, subcapsular epithelium of thymus, follicular dendritic reticulum cells, skeletal muscles, epithelium of lens, and glial cell fibers in the central nervous system in early embryos. In malignant tumors, MN-4 immunostaining was consistently present in all melanomas, angiosarcomas, Kaposi's sarcomas, leiomyosarcomas, mucoepidermoid carcinomas of salivary gland, placental-site trophoblastic tumors, and choriocarcinomas. Three of 8 squamous cell carcinomas, 2 of 2 small cell carcinomas of the lung, 2 of 11 of infiltrating breast carcinomas, and 4 of 11 of germinomas were focally and weakly positive for MN-4 antibody. In contrast, a wide variety of other carcinomas, sarcomas, lymphomas, leukemias, and neuroendocrine tumors failed to show MN-4 immunoreactivity. In conclusion, MN-4 is a specific monoclonal antibody that recognizes Mel-CAM on paraffin sections. This antibody is useful in retrospective studies of Mel-CAM expression in archival tissue sections and might provide a diagnostic and prognostic marker for human neoplasms.

Animals↗
PubMed1997

[Study of detecting p53 gene of laryngeal carcinoma on paraffin-embedded tissue sections by in situ polymerase chain reaction].

In order to investigate alterations of p53 gene in laryngeal carcinoma, five cases of paraffin-embedded tissue were detected by in situ polymerase chain reaction (PCR). The results showed that p53 gene was presented in more than 90% of cancer cells in all cases with exon 5 specific primers, which signal copy numbers were 2.24 +/- 0.23. It suggests that in situ PCR and paraffin-embedded tissue are able to be used in the single copy gene research. The improvement and application of the technique were discussed.

Carcinoma, Squamous Cell↗
PubMed2005

[Detection of FUS-CHOP fusion gene in paraffin-embedded tissues and its clinicopathologic significance for myxoid/round cell liposarcomas].

OBJECTIVE: To explore the feasibility of detecting FUS-CHOP fusion gene in formalin-fixed, paraffin-embedded tissue and its application in the diagnosis and differential diagnosis of myxoid/round cell liposarcomas (MRCLs). METHODS: Forty-four formalin-fixed, paraffin-embedded MRCL samples and 60 control cases (atypical/well-differentiated liposarcoma, pleomorphic liposarcoma, low-grade myofibrosarcoma, etc.) retrieved from the archival files were studied. Nested reverse transcription-polymerase chain reaction (RT-PCR) technique was employed to detect the FUS-CHOP mRNA expression, followed by DNA sequencing confirmation of the PCR product. Housekeeping gene PGK was used to assess the quality of the mRNA templates. RESULTS: PGK mRNA was detected in 93 of 104 tumor cases (89.4%), including 39 MRCLs cases (39/44, 88.6%) and 90% of the negative control cases. Type II FUS-CHOP fusion transcript was successfully detected in 20 out of 39 (51.3%) MRCL cases. Type I FUS-CHOP fusion transcript was not detected in any MRCLs in this study. All 60 negative control cases were negative for the FUS-CHOP fusion gene transcripts. CONCLUSIONS: (1) Nested RT-PCR can be used to detect FUS-CHOP mRNA in formalin-fixed, paraffin-embedded tissues. (2) FUS-CHOP is considered a specific molecular and genetic hallmark for MRCLs. Nested RT-PCR is a sensitive and specific technique in detecting FUS-CHOP gene, and can be used in the diagnosis and differential diagnosis of MRCLs.

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↗
PubMed1993

Identification of Toxoplasma gondii in formalin-fixed, paraffin-embedded tissue by polymerase chain reaction.

We investigated the use of polymerase chain reaction (PCR) for diagnosis of toxoplasmosis in formalin-fixed, paraffin-embedded tissue, using seven cases in which the diagnosis was certain based on histologic examination and eight cases in which histologic findings only suggested Toxoplasma gondii infection. After amplification the PCR products were subjected to agarose gel electrophoresis followed by Southern blotting with a 32P-labeled oligonucleotide probe. In six of seven proven cases of toxoplasmosis, organisms were detected using DNA extracted from 1/50 to 3/50 of a 6-microns tissue section. In addition, organisms were detected in one of eight cases in which infection was suspected, but not proven by identification of organisms. We conclude on the basis of this study that PCR may be useful for detection of T. gondii in a few cases in which routine histologic examination or immunoperoxidase staining is not definitive. Since PCR has 1000-fold higher sensitivity when applied to fresh, rather than paraffin-embedded, tissues, we expect PCR to be more sensitive in fresh tissue.

Animals↗
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↗
PubMed2003

[Detection of cyclin D1 protein expression and t(11;14) chromosomal translocation in paraffin-embedded tissues and its clinicopathologic significance for mantle cell lymphoma].

OBJECTIVE: To investigate the feasibility of detecting cyclin D1 protein expression and t(11;14) chromosomal translocation in paraffin-embedded tissues and its diagnostic and differential diagnostic significance for mantle cell lymphoma (MCL). METHODS: Paraffin-embedded samples of 36 cases of MCL and a control group of 71 cases of small B-cell lymphomas were retrieved from archive materials. Immunohistochemical staining for cyclin D1 and semi-nested PCR for t(11;14) were detected in all samples. House-keeping gene beta-actin was used to detect the quality of DNA. RESULTS: (1) Cyclin D1 was expressed in 26 of the 36 MCL (72.2%). There was no cyclin D1 expression in the control group. (2) beta-actin DNA was detected in 101 of the 107 tumor cases (94.4%). t(11;14) was detected in 22 of the 36 MCL. Translocation was not found in control group. The positive rate for t(11;14) was 64.7% in MCL after exclusion of 2 cases which were negative for both t(11;14) and beta-actin. (3) 29 cases were positive for cyclin D1 and/or t(11;14), the positive rate reached 80.5%. CONCLUSION: The combined detection of cyclin D1 and t(11;14) in paraffin-embedded tissues is found to be a specific and feasible method for diagnosis and differential diagnosis of mantle cell lymphoma.

Adult↗
PubMed1989

In situ hybridization using PEG-embedded tissue and riboprobes: increased cellular detail coupled with high sensitivity.

We describe a procedure for preparing tissue sections by embedding in polyethylene glycol for subsequent in situ hybridization analysis using single-stranded RNA probes. Improved tissue morphology is obtained as compared to frozen sections, and the embedding procedure is milder and faster than paraffin embedding. Sections as thin as 2 microns are readily cut from PEG-embedded brain tissue. A simplified hybridization protocol (Clayton et al.: Neuron 1:249, 1988) supports the detection of even low-abundance brain mRNAs (less than or equal to 10(-4) fractional mRNA mass). By employing high stringency washes in place of ribonuclease treatment after hybridization, cell RNA is retained for cresyl violet staining, and high signal:noise ratios are achieved. Solutions to problems with section mounting and adherence to glass slides are presented. The combination of improved morphology, high signal levels, and relative simplicity should make this procedure useful in a variety of applications.

Animals↗
PubMed2004

Microdissected region-specific gene expression analysis with methacarn-fixed, paraffin-embedded tissues by real-time RT-PCR.

We have previously shown methacarn to be a versatile fixative for analysis of proteins, DNA, and RNA in paraffin-embedded tissues (PETs). In this study we analyzed its suitability for quantitative mRNA expression analysis of microdissected PET specimens using a real-time RT-PCR technique. Fidelity of expression in the methacarn-fixed PET sections, with reference to dose-dependent induction of cytochrome P450 2B1 in the phenobarbital-treated rat liver, was high in comparison with the unfixed frozen tissue case, even after hematoxylin staining. RNA yield from methacarn-fixed PET sections was equivalent to that in unfixed cryosections and was also not significantly affected by hematoxylin staining. Correlations between the expression levels of target genes and input amounts of extracted RNA in the range of 1-1000 pg were very high (correlation coefficients >0.98), the regression curves being similar to those with unfixed cryosections. Although cell numbers should be optimized for each target gene/tissue, >/=200 cells were necessary for accurate measurement in 10-microm-thick rat liver sections judging from the variation of measured value in small microdissected areas. These results indicate high performance with methacarn, close to that of unfixed tissues, regarding quantitative expression analysis of mRNAs in microdissected PET-specimens.

Acetic Acid↗