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PubMed2003

[Amplification of beta-catenin gene in the paraffin-embedded epidermal tissue].

OBJECTIVE: To establish a method of extracting DNA from the paraffin-embedded epidermal tissue and examine the amplification of beta-catenin gene by polymerase chain reaction (PCR) analysis. METHODS: Exon 3 of the beta-catenin gene was amplified by PCR in 14 paraffin-embedded Bowen's disease (BD) of various weight and years. RESULTS: The amplification of exon 3 of beta-catenin gene was successful in all the 10 mg and 20 mg samples, but not in the 5 mg samples. The amplification of exon 3 of beta-catenin gene was successful in 9 samples embedded within 10 years, and in only 2 of the 5 samples embedded more than 10 years. CONCLUSION: Epidermal tissue whose weight is 10 mg or more within 10 years of embedding can be used in the amplification of purpose gene by PCR.

Cytoskeletal Proteins↗
PubMed1994

Immunohistochemical staining for DNA topoisomerase II in frozen and formalin-fixed paraffin-embedded human tissues.

DNA topoisomerase II is the molecular target of several clinically useful chemotherapeutic drugs. The sensitivity of cells to drugs that target topoisomerase II is dependent on the cellular content of this enzyme. Drug-sensitive cells have elevated amounts of type II topoisomerase. To determine relative amounts of enzyme in malignant neoplasms, we developed an in situ immunohistochemical stain for topoisomerase II. The stain uses either polyclonal or monoclonal antibodies produced against the alpha isoform of the enzyme. Staining can be done on both frozen and formalin-fixed, paraffin-embedded tissues. By using this immunostain, we found marked differences in enzyme content in several human malignancies.

Animals↗
PubMed2006

Direct cancer tissue proteomics: a method to identify candidate cancer biomarkers from formalin-fixed paraffin-embedded archival tissues.

Successful treatment of multiple cancer types requires early detection and identification of reliable biomarkers present in specific cancer tissues. To test the feasibility of identifying proteins from archival cancer tissues, we have developed a methodology, termed direct tissue proteomics (DTP), which can be used to identify proteins directly from formalin-fixed paraffin-embedded prostate cancer tissue samples. Using minute prostate biopsy sections, we demonstrate the identification of 428 prostate-expressed proteins using the shotgun method. Because the DTP method is not quantitative, we employed the absolute quantification method and demonstrate picogram level quantification of prostate-specific antigen. In depth bioinformatics analysis of these expressed proteins affords the categorization of metabolic pathways that may be important for distinct stages of prostate carcinogenesis. Furthermore, we validate Wnt-3 as an upregulated protein in cancerous prostate cells by immunohistochemistry. We propose that this general strategy provides a roadmap for successful identification of critical molecular targets of multiple cancer types.

Amino Acid Sequence↗
PubMed1993

Characterization of anti-HLA class II monoclonal antibody LGII-612.14 reacting with formalin fixed tissues.

mAb LGII-612.14 derived from a BALB/c mouse immunized with interferon-gamma (IFN-gamma) treated cultured human B lymphoid cells LG-2 has been shown with serological and immunochemical assays to recognize a monomorphic determinant expressed on the beta chain of HLA-DR, -DQ and -DP antigens. The linear nature of the determinant, which is likely to be formed by residues 19-25, is indicated by the reactivity of mAb LGII-612.14 with HLA-DR, -DQ and -DP beta chains purified by electrophoresis in presence of SDS. An unusual characteristic of mAb LGII-612.14 is its reactivity with fixed tissue sections. The intensity of staining is affected by the incubation temperature, the incubation time and the fixative used. Maximal intensity of staining of formalin fixed, paraffin embedded tissue sections required an incubation time of 16 h. The intensity of staining of paraffin embedded tissues initially fixed with Bouin's solution, formalin or ethanol was similar to that of frozen tissue sections and stronger than that of tissues fixed with B5 solution. No staining was detected of paraffin embedded tissues fixed with glutaraldehyde or Zenker's solution. Comparison of the staining patterns with mAb LGII-612.14 of frozen and fixed tissue sections showed that the latter substrates provide a superior detail of tissue architecture and cellular morphology without significant loss of sensitivity. Furthermore, comparison of the characteristics of mAb LGII-612.14 with the few previously published anti-HLA class II mAb reacting with fixed tissues indicates that mAb LGII-612.14 stains formalin fixed, paraffin embedded tissues, while mAb 910D7 and TAL-1B5 stain tissues fixed with less commonly used fixatives. Furthermore, mAb LGII-612.14 is likely to yield more sensitive staining results than anti-HLA-DR, -DQ and -DP mAb KUL/05. The present results indicate that mAb LGII-612.14 represents a useful probe to apply immunohistochemical techniques to the analysis of the distribution of HLA class II antigens in fixed tissues. This will greatly facilitate the use of readily available collections of fixed tissue specimens in retrospective studies to assess the clinical significance of changes in HLA class II antigen expression which occur in various disease states.

Animals↗
PubMed1993

Improved DNA content histograms from formalin-fixed, paraffin-embedded liver tissue by proteinase K digestion.

An improved method for the enzymatic digestion of formalin-fixed, paraffin-embedded liver tissue for DNA content analysis by flow cytometry is presented. Forty samples of histologically normal liver were alternately digested by the traditional pepsin method or a new method utilizing proteinase K and heat. Sixteen (40%) of the pepsin-digested samples had apparent DNA aneuploid peaks by flow cytometry. False DNA aneuploid peaks were not present in any of the histograms obtained after proteinase K digestion. Microscopy showed that the pepsin-digested samples had residual cytoplasmic remnants which contained fluorescent material. Samples digested with proteinase K had few cytoplasmic remnants. The average G0/G1 coefficient of variation after proteinase K treatment was lower (41%) and the fluorescent intensity higher (128%) than the pepsin-treated samples. The apparent mean S-phase (a combination of S-phase cells and underlying debris) after proteinase K digestion was 35% of the pepsin-treated samples. Primary and secondary tumors of the liver that were DNA aneuploid after pepsin treatment were also DNA aneuploid after proteinase K treatment. A modified digestion protocol utilizing proteinase K and heat can provide superior results for DNA content analysis of formalin-fixed, paraffin-embedded liver tissue.

Aneuploidy↗
PubMed1982

An improved method for HBcAg demonstration in paraffin-embedded liver tissue.

Factors influencing the preservation of HBcAg in formalin-fixed, paraffin-embedded liver tissue have been analysed. Fixation time, choice of the embedding medium, deparaffinization and selection of anti-HBc proved to be critical variables. For optimal results, fixation in 4% buffered formalin for no longer than 24-48 h, the use of wax with a low melting point, a heating step prior to deparaffinization, and a search for anti-HBc specifically reactive for paraffinated HBcAg are recommended.

Fixatives↗
PubMed1985

Generation of monoclonal antibodies recognizing neuronal elements in formalin-fixed paraffin-embedded human tissue.

We used formalin-fixed human spinal cord and dorsal root ganglia as immunogens to generate monoclonal antibodies (mAb) which immunohistochemically react with neurons in formalin-fixed human tissue sections. Three of the mAb recognized all neuronal populations studied, including those in spinal cord, dorsal root ganglia, cerebellum, and cerebrum. A fourth mAb recognized neurons within spinal cord, dorsal root ganglia and dentate nucleus of cerebellum but not those in cerebrum or cerebellar hemispheres. This mAb, unlike the other three, did not recognize murine neurons. These data indicate the feasibility of generating mAb suitable for analysis of human pathological material in its most readily available form, formalin-fixed paraffin-embedded tissue.

Antibodies, Monoclonal↗
PubMed1994

A retrospective study of DNA ploidy of transitional cell carcinoma of urinary bladder using flow cytometry.

Transitional cell carcinoma is the most common malignancy of urothelial origin in urinary bladder. Its aggressive potential cannot be reliably predicted from the histologic appearance alone. Recently, there has been extensive studies of DNA ploidy using flow cytometry in prospective analysis of bladder tumors with fresh tissue. Techniques for the preparation of paraffin embedded tissue have been developed and successfully applied in retrospective evaluation of bladder tumors. These studies had demonstrated the correlation between histological grading and DNA ploidy, and between clinical staging and DNA ploidy. Our retrospective study showed that the cytometric method is superior to histologic grading in the predictability of patient's survival, especially in the low grade (grades 1 and 2) group. In the preparation of paraffin embedded tissue, the high background debris and wide range of coefficients of variation had some interference in the interpretation for the histogram of FCM (only 80% of the cases studied are interpretable). This interference might be due to the variation of conditions of tissue processing and fixation. The evaluation of S phase fraction and G2/M fraction is also difficult to perform because of the same problem. There was an encouraging result in the evaluation of DNA ploidy using flow cytometry with paraffin embedded tissue, although a relatively small number of patients were included in this study. The value of this technique application in clinical evaluation of the prognosticity was obvious.

Adult↗
PubMed1999

Y chromosome enumeration in touch preparations from 42 prostate tumors by interphase fluorescence in situ hybridization analysis.

A change in Y chromosome number is but one of the many cytogenetic abnormalities reported in human prostate tumors. However, reports in the literature have varied regarding the frequency of Y loss or gain, whether it is restricted to the cancerous tissue, and its relation to the biology of the disease. The most frequently used materials for analysis of Y enumeration have been metaphase spreads from short-term cell cultures of prostate tumor tissue and paraffin-embedded tissue sections. Analysis of Y chromosome number by using metaphase spreads on short-term cultures can be misleading owing to clonal cell selection during the establishment of these cultures. This may result in an incomplete representation of the loss/gain pattern in the tumor as a whole. Studies using paraffin-embedded tissue sections can be complicated by apparent chromosome loss due to nuclear truncation as a result of tumor sectioning. In an attempt to circumvent these problems, we have used touch preparations from human prostate tumors to search for Y chromosome loss. Fluorescence in situ hybridization analysis was conducted by using a whole chromosome Y paint, with an alpha-satellite chromosome 3 probe as a control, on tumor samples from 42 patients ages 40-75. The results demonstrated a gain of Y in a single prostate tumor sample, with no convincing evidence for loss of the entire Y chromosome in any of the other 41 samples examined. The results suggest that loss of the entire Y chromosome is an infrequent event in prostate cancer.

Adult↗
PubMed2003

A simple and reliable pretreatment protocol facilitates fluorescent in situ hybridisation on tissue microarrays of paraffin wax embedded tumour samples.

AIMS: To describe a robust pretreatment protocol for preparing paraffin wax embedded tissues on tissue microarrays for fluorescence in situ hybridisation (FISH). The newly developed pretreatment protocol described here was compared with the commonly used sodium thiocyanate based protocol and two different heating methods used in standard antigen unmasking protocols for immunohistochemistry (pressure cooking and microwaving in citrate acid buffer). METHODS: Dewaxed tissue sections were incubated in 10mM citric acid buffer at 80 degrees C for 30 minutes to two hours, followed by a short pepsin digestion (1-5 mg/ml). Pretreated tissues were co-denatured with DNA probes at 80 degrees C for 10 minutes, followed by hybridisation at 37 degrees C for 48-72 hours. RESULTS: The three protocols using citrate acid buffer produced FISH signals with superior signal to noise ratios compared with sodium thiocyanate pretreatment. Most importantly, the best tissue attachment was achieved using the newly developed pretreatment protocol: on tissue microarrays less than 1% of cores were lost. To date, a total of 30 probes have been successfully hybridised on to breast tissue and multi-tissue microarrays. CONCLUSION: This pretreatment protocol is easy, reproducible, and facilitates FISH on tissue microarrays, with potential for widespread application in cancer research.

Breast Neoplasms↗
PubMed2007

The heterogeneous distribution of monosomy 3 in uveal melanomas: implications for prognostication based on fine-needle aspiration biopsies.

CONTEXT: The detection of monosomy 3 in uveal melanomas has repeatedly been associated with adverse outcome. Fine-needle aspiration biopsy is being used to detect monosomy 3 in these tumors, based on the assumption that this chromosomal abnormality is distributed homogeneously throughout the tumor. OBJECTIVE: To study the distribution of monosomy 3 in primary uveal melanoma by fluorescence in situ hybridization (FISH). DESIGN: We studied 50 enucleated eyes with uveal melanoma. In all 50 tumors we performed cytogenetic analysis and FISH using a DNA-specific probe for the centromere region of chromosome 3 on cultured tumor cells. In addition, the percentage of tumor cells with monosomy 3 was assessed by FISH on nuclei, isolated from paraffin-embedded tissue and compared to results of FISH on regular histology sections of the paraffin-embedded tissue. RESULTS: Combining karyotyping and FISH on cultured cells identified monosomy 3 in 19 (38%) of 50 tumors, whereas FISH on nuclei isolated from paraffin-embedded tissue showed 31 (62%) of 50 as having monosomy for chromosome 3. FISH analysis on paraffin sections showed tumor heterogeneity for copy number of chromosome 3 in at least 7 cases. CONCLUSIONS: FISH analysis on paraffin sections shows that heterogeneity of monosomy of chromosome 3 is a frequent phenomenon in uveal melanoma. FISH on nuclei isolated from paraffin-embedded tissue identifies a higher frequency of monosomy 3 than the traditional combination of karyotyping and FISH on cultured uveal melanoma cells. The practice of assigning patients to risk categories based on fine-needle aspiration biopsy samples from primary uveal melanoma may be subject to error based on the heterogeneous distribution of monosomy 3 in these tumors.

Adult↗
PubMed1998

Detection of Mycobacterium tuberculosis in paraffin embedded intestinal tissue specimens by polymerase chain reaction: characterization of IS6110 element negative strains.

Tuberculosis is still one of the most widespread infection known to mankind. Although lung is the predominant site of disease, a sizeable population in Pakistan gets intestinal disease. Clinical presentation, radiologic and endoscopic examination provide clues to the diagnosis. However, a definitive diagnosis requires biopsy material with granulomas and/or caseation complemented by acid fast staining and culture. There are many occasions when biopsy material is scanty and even in some intestinal resection cases histologic evaluation fails to confirm or rule out tuberculosis. Therefore, an investigation was conducted to assess the efficacy of PCR in the detection of mycobacterial DNA in paraffin embedded intestinal tissue. In this study 12 histologically confirmed cases of intestinal tuberculosis and 2 cases with non specific inflammation but clinically suspected for abdominal tuberculosis were selected. One case of rectal polyp was included to serve as a negative control. M. tuberculosis DNA was amplified in 8 out of 12 histologically confirmed cases and in 2 cases diagnosed with non specific inflammation. Amplified products were obtained in 6 out of 10 PCR positive specimens with IS6110 region specific primers while 4 samples were negative, suggesting the absence of insertion sequence 6110 in these strains. However, amplification was obtained in these negative specimens with a second primer pair confirming them as M. tuberculosis complex species. On the basis of this study we conclude that; (1) Processed and paraffin embedded tissue material is suitable for PCR analysis, (2) PCR assay can be used to complement the diagnosis of intestinal tuberculosis especially in situations where a definite conclusion can not be drawn by conventional methods, (3) M. tuberculosis species lacking insertion sequence 6110 element are present in our population. Therefore, several primer pair sets should be included when applying PCR for the detection of mycobacterial DNA.

Adult↗
PubMed1999

Loss of heterozygosity at 3p in benign lesions preceding invasive breast cancer.

BACKGROUND: Loss of heterozygosity (LOH) at chromosome 3p is one of the most common genetic abnormalities identified in human cancers and has occasionally been noted in benign proliferative lesions predisposing to breast cancer. If the frequency of LOH at 3p in benign proliferative lesions correlates with the subsequent development of breast cancer, it may be possible to develop powerful tools for molecular risk assessment based on this technology. MATERIALS AND METHODS: Archival paraffin-embedded tissues from benign breast biopsies in five women who have developed breast cancer and three women who have not developed breast cancer were microdissected and allelotyped at 3p using six microsatellite markers. RESULTS: No LOH was detected in the biopsies from women who have not developed breast cancer. For women developing breast cancer, the proportion of informative loci showing LOH in the benign proliferative lesions was 0.47 as compared to 0.57 for the associated breast cancers. There was no LOH detected in epithelial DNA from a fibroadenoma. Of 15 informative loci, 4 (27%) showed LOH in both the benign proliferative lesion and the associated cancer; however, the actual parental allele lost was different in three of these four cases. CONCLUSIONS: These results suggest that there are specific patterns of genetic instability common to preneoplastic lesions and the breast cancers that subsequently develop even when the paired lesions are not clonally related. LOH analysis of benign breast epithelium may provide a tool for molecular risk assessment and a surrogate endpoint for breast cancer chemoprevention trials.

Adult↗
PubMed1993

Adaptation of a non-radioactive in situ hybridization method to electron microscopy: detection of tenascin mRNAs in mouse cerebellum with digoxigenin-labelled probes and gold-labelled antibodies.

In this study we describe a method for the detection of mRNAs at the ultrastructural level using a non-radioactive in situ hybridization method based on digoxigenin-labelled cRNA probes and gold-labelled digoxigenin-specific antibodies. We applied this protocol to an analysis of the expression of the extracellular matrix protein tenascin in the developing cerebellar cortex of the mouse. To gain an impression of the sensitivity attainable with digoxigenin-labelled probes, we first established at the light microscopic level that the hybridization signal obtained with the non-radioactive probe is as sensitive as that obtained with a 35S-labelled probe. The non-radioactive hybridization protocol was then combined with electron microscopic post-embedding and immunogold detection techniques. Tenascin-specific, digoxigenin-labelled cRNA probes were hybridized to ultrathin sections of Lowicryl K4M-embedded tissue and the probe/target mRNA hybrids were detected using gold-labelled antibodies to digoxigenin. In agreement with the observations from in situ hybridization at the light microscopic level, specific labelling was observed in Golgi epithelial cells in the region of the Purkinje cell layer and cells in the internal granular layer, which could be identified as astrocytes by ultrastructural criteria. Labelling was detectable in association with free ribosomes and ribosomes of the rough endoplasmic reticulum. In addition, focal hybridization signals were occasionally found in the nucleus. No signal was observed in Golgi epithelial cells or astrocytes using sense or in any other cerebellar cell type using either sense or anti-sense probes. The described in situ hybridization technique uses ultrastructural criteria to associate the presence of a given mRNA species with a particular cell type. Additionally, it provides information about the target mRNA's subcellular distribution, thus offering the possibility to study intracellular transport of particular mRNAs.

Acrylic Resins↗
PubMedDate not supplied

Immunohistochemical detection of E-cadherin, alpha- and beta-catenins in papillary thyroid carcinoma.

E-cadherin and catenins play a major role in neoplastic cell behavior as a suppressor of invasion and/or metastasis. The aim of this study was to determine E-cadherin, alpha-catenin and beta-catenin expressions in papillary thyroid carcinoma (PTC) and to correlate the results of expression to initial clinicopathological parameters and clinical outcome. Forty-one cases (mean age 37.3 +/- 11.2 yr) with PTC were studied. Patients were followed-up with a mean period of 47.6 +/- 27.0 months. A retrospective immunohistochemical analysis of E-cadherin, alpha-catenin and beta-catenin was performed on paraffin-embedded tissue sections. Tissues from ten patients with benign goiter were used as controls. E-cadherin, alpha- and beta-catenin immunoreactivities were found in 80% (33/41), 76% (31/41) and 97% (40/41) of patients respectively. No correlation was found between E-cadherin, alpha- and beta-catenin immunoreactivities and sex, local invasion or lymphatic spread at the time of initial examination. Distant metastases and/or local recurrences developed in 6 patients during follow-up. Recurrences/metastases developed both E-cadherin, alpha- and beta-catenin positive and negative primary tumors. Disease-free survival curves according to Kaplan-Meier analysis and log-rank test did not show any significant differences between E-cadherin, alpha- and beta-catenin positive and negative patients. According to our findings, E-cadherin, alpha- and beta-catenin expressions may not add any valuable information to the follow-up in a subgroup of PTC patients with a relatively benign course.

Adult↗
PubMed2000

Quality assessment in diagnostic molecular pathology: experience from a German-Austrian-Swiss multicenter trial.

In order to assess the current technical standard of diagnostic molecular pathology, we have conducted a multicenter trial with 34 participating pathology laboratories in Germany, Austria and Switzerland. Formalin-fixed, paraffin-embedded tissue blocks were selected from 15 cases, comprising 4 B-cell non-Hodgkin's lymphomas, 4 T-cell non-Hodgkin lymphomas, 4 cases with lymphadenitis, 2 cases with confirmed tuberculosis and 1 case of sarcoidosis. All participating laboratories received one 10-microm section from each of the 15 cases to detect clonality using immunoglobulin heavy chain (IgH) gene or T-cell receptor (TCR)-gamma gene rearrangement analysis in 12 and mycobacterial DNA in 3 cases. In addition, participants had to answer technical questions about the application of internal quality controls and performance of fragment length or sequence analysis. Correct results were reported in 80% and 90% for IgH and TCR-gammagene rearrangement analysis, respectively, and in 83% for mycobacterial DNA analysis. No significant differences in the quality of results were obvious when the individual techniques used for molecular analysis were compared. However, when two independent techniques were used by the same laboratory, a higher rate of correct results was obtained for IgH and TCR rearrangement analysis. In conclusion, this study demonstrates a high technical standard of molecular diagnostic adjuncts among the participating laboratories. Regular multicenter trials with a greater number of participating laboratories working in this field will be indispensable to ensure a continuing or increasing standard in diagnostic molecular pathology.

Austria↗
PubMed1983

Congenital blood cysts of the heart valves.

Congenital blood cysts of the heart valves are found most commonly on the tricuspid and mitral valves of fetuses and infants. Hearts available following 38 random autopsies of fetuses and infants 2 years of age or younger were examined. Blood cysts were found in 18 cases (47 per cent) in which ages ranged from 26 weeks of gestation to 11 months. The cysts varied in diameter, from microscopic to 3 mm. Affected valves had from one to 20 cysts. Light microscopic examination of serially sectioned paraffin-embedded tissue and plastic-embedded tissue and scanning electron microscopic examination revealed connections between the cyst lumens and ventricles via small endothelium-lined channels. The cyst structure suggested formation from ventricular endothelial infoldings in the valve leaflet base, which bulged into the atrium because of the pressure gradient present during valve closure. Blood cysts are a common finding in neonates dying of various causes and probably have no clinical significance. There is no association with asphyxia as previously described. Blood cysts may persist and enlarge to form giant cysts of the heart valves.

Aortic Valve↗