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Results for “Paraffin Embedding”

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At least 181 records · Page 10Linked to original sources

PubMed1994

DNA content in non-Hodgkin's lymphoma. Comparison between flow cytometry and cytogenetics in fresh and paraffin-embedded tissue.

DNA content of 36-non-Hodgkin's lymphomas was analyzed by flow cytometry (FCM) and cytogenetics (CG), 21 in fresh and 15 in paraffin-embedded tissue. The results of both techniques were coincident in 60% of the fresh tissue samples and in 45% of the paraffin-embedded ones, the reason for this difference could be the poor resolution of DNA histograms from paraffin-embedded tissue. All samples judged as aneuploid by FCM were aneuploid also by CG. Some samples with a hyperdiploid population by CG gave a diploid population by FCM with a 'false' high DNA-synthesis (S) fraction. From a technical point of view, CG and FCM have to be performed on the same fresh tissue.

Cell Cycle↗

PubMed1993

Determination of B-cell clonality in paraffin-embedded lymph nodes using the polymerase chain reaction.

Formalin-fixed, paraffin-embedded tissue from B-cell malignant lymphomas (26), reactive lymphadenopathies (8), non-B-cell malignancies (5), and atypical lymphoproliferative lesions (7) were analyzed for clonal immunoglobulin heavy chain gene rearrangement by the polymerase chain reaction (PCR), using consensus primers for the variable and joining regions of the gene. By employing a high-resolution gel electrophoresis technique, we were able to demonstrate one or two dominant bands, indicating a clonal population, in 15 of the 23 cases (65%) of B-cell lymphoma in which amplification occurred. Six of six reactive lymph nodes in which amplification occurred produced a multi-banded pattern indicative of a polyclonal population. This improved PCR technique allows a clearer distinction between clonal and polyclonal patterns than other previously proposed methods. It also works well in paraffin-embedded tissue and may therefore be a useful adjunct to the diagnostic armamentarium applied to archival material.

Base Sequence↗

PubMed1995

[Histological processing of iliac crest biopsies based on decalcification and paraffin embedding with reference to osteolytic and hematologic diagnosis].

A survey is given of methods involving decalcification and paraffin embedding of iliac crest biopsy for osteological and haematological diagnostic procedures. In order to avoid shrinkage, loss of antigens, and fading of ferritin iron and enzymes, a fixative has been designed that is composed of an aqueous solution of calcium acetate (10(-1) M), glutaraldehyde (0.5%), and formaldehyde (1%; CGF). CGF-fixated specimens are decalcified in an aqueous solution of 10% di-sodium ethylene-diaminotetraacetate (EDTA) neutralized by tris[hydroxy]methylaminomethane and embedded in paraffin. Tissue prepared in this manner allows histochemical detection of naphthol AS-D chloroacetate esterase in the neutrophilic cell line and in tissue mast cells, tartrate-resistant acid phosphatase in hairy cells and certain other low malignant B-cell lymphomas, in Gaucher cells, and in osteoclasts, and a specific platelet esterase in megakaryocytes and leukaemic megakaryoblasts. A broad panel of antigens is well preserved. Beside haemosiderin, cytosolic ferritin can be detected by Perls' reaction in acute phase-stimulated macrophages. Emphasis is placed on the diagnostic impact of plasma cell siderosis and lysosomal sideroblastocytosis in haemochromatosis and in alcoholism respectively. A technique is presented to discriminate mineralized and non-mineralized bone even after decalcification.

Bone Diseases↗

PubMed1993

The identification of equid herpesvirus 1 in paraffin-embedded tissues from aborted fetuses by polymerase chain reaction and immunohistochemistry.

Paraffin-embedded organ samples from 28 aborted fetuses and three foals, partly archival and partly sampled in 1991, were examined by polymerase chain reaction (PCR) and immunohistochemistry for the presence of DNA and antigens, respectively, specific for equine herpesvirus 1 (EHV-1). Virologic examination had been performed on 23 of the aborted fetuses. DNA fragments specific for EHV-1 were identified by PCR, and EHV-1 antigens were identified in situ by immunohistochemistry, with an agreement between the methods of 94% (kappa = 0.85). Compared with virus isolation, PCR agreement was 87% (kappa 0.69), and IH agreement was 82% (kappa = 0.47). These results showed that there was moderate to almost perfect agreement among the different methods and that PCR and immunohistochemistry are powerful tools for the identification of EHV-1 in paraffin-embedded tissues. The techniques give more rapid results than virus isolation and also detect inactivated virus, which are not identified by standard virus isolation. These techniques also make retrospective studies possible.

Abortion, Veterinary↗

PubMed1993

p53 protein immunostaining in routinely processed paraffin-embedded sections.

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human tumors. The altered protein product of the mutant gene is stabilized in tumor cells and can be detected using monoclonal antibody immunohistochemistry. We report a technique for immunostaining of the altered p53 protein in routinely processed paraffin-embedded tissue sections, comparing this method with a frozen-section immunostain method for concordance. Similarly processed tumor cell lines with known mutations or deletions of the p53 gene served as positive or negative controls. The results in 25 human gliomas and 20 colorectal carcinomas showed a 100% correspondence of positive reactivity in the colorectal carcinomas and an 83% correspondence in the gliomas. In no case did the paraffin-embedded sections react in the absence of frozen section reactivity (that is, there were no false positives). Three of 18 gliomas showed reactivity in frozen sections without reactivity in the companion paraffin-embedded sections. This discrepancy could have been caused by technical factors such as length of fixation time. We propose that this new paraffin-embedded section immunostaining method will be of value as a screening technique for the investigation of p53 mutations in archived human tumors. The data thus obtained may then be correlated with clinical information and perhaps be of value in diagnosis or predicting outcome for various human cancers.

Adenocarcinoma↗

PubMed1989

Comparison of flow cytometric data obtained using fresh and paraffin-embedded lymphoid tissue.

Flow cytometric (FCM) DNA analysis was carried out on 24 lymph nodes: 13 from benign reactive hyperplasias and 11 from non-Hodgkin's lymphomas. FCM was performed on two types of samples: (1) fresh cell suspensions and (2) suspensions prepared from formalin-fixed, paraffin-embedded sections. FCM of fresh samples detected aneuploidy in 23 of the 24 cases while FCM of paraffin-embedded samples detected aneuploidy in only 6 of the 24 cases. Those six cases were lymphomas considered histologically as having a poor prognosis. Only one case, a lymphoma, was euploid with both methods. The coefficients of variance determined in each case for both methods were found to be within "normal ranges," but were greater in the paraffin-embedded specimens. The results suggest that FCM DNA analysis of formalin-fixed, paraffin-embedded sections does not have as great a resolution capacity as does analysis of fresh cell suspensions, since the former failed to detect cell populations that had a small degree of aneuploidy (close to the 2n population).

DNA↗

PubMed2005

JC virus genotyping using formalin-fixed, paraffin-embedded renal tissues.

Recently genotyping of JC virus (JCV) DNA in renal tissue was reported to be useful to identify the geographic origin of unidentified cadavers. In the above study, autopsied tissue samples without storage or stored in a frozen state were used. This study examined JCV DNA sequence modifications caused by formalin-fixation, in an attempt to elucidate whether formalin-fixed, paraffin-embedded tissue samples can also be used to determine the genotypes of JCV DNA in the kidney. In four cases, a 610 bp typing region of the JCV genome was PCR-amplified from renal tissues stored for 1 year in three different states: frozen at -80 degrees C [Amaker, B.H., Chandler, F.W., Huey, L.O., Colwell, R.M., 1997. Molecular detection of JC virus in embalmed, formalin-fixed, paraffin-embedded brain tissue. J. Forensic Sci., 1157-1159], formalin-fixed, paraffin-embedded [Ault, G.S., Stoner, G.L., 1992. Two major types of JC virus defined in progressive multifocal leukoencephalopathy brain by early and late coding region DNA sequences. J. Gen. Virol. 73, 2669-2678], and soaked in 5% formalin [Baksh, F.K., Finkelstein, S.D., Swalskey, P.A., Stoner, G.L., Ryschkewitsch, C.F., Randhawa, P.R., 2001. Molecular genotyping of BK and JC virus in human polyomavirus-associated interstitial nephritis after renal transplantation. Am. J. Kidney Dis. 38 (2), 354-365]. The amplified fragments were cloned, and the resultant clones were sequenced. In frozen samples, single sequences ('original' sequences) were detected in all cases. In formalin-fixed, paraffin-embedded samples, not only the original sequences but also those with 1-6 base substitutions were detected. From formalin-soaked samples, the original sequences and those with 1-5 and 10-13 substitutions were detected. The genotyping of JCV DNA was not hampered by the presence of 1-6 substitutions, but a shift in JCV genotypes was observed in sequences with 10-13 substitutions. Thus, it was concluded that the genotypes of JCV DNA in the kidney can be determined only with specimens stored in a frozen state or formalin-fixed for a short time.

Adult↗

PubMed2004

[Detection of EWS-FLI1 fusion transcript in Ewing's sarcoma/peripheral primitive neuroectodermal tumors by one-step RT-PCR using paraffin-embedded tissues].

OBJECTIVE: To investigate the expression of EWS-FLI1/ERG fusion transcript resulting from t(11;12)(q24;12) in paraffin-embedded tissues and its diagnostic implication for Ewing's sarcoma/peripheral primitive neuroectodermal tumors (ES/pPNET). METHODS: One-step reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to detect a characteristic EWS-FLI1/ERG fusion transcript in 25 cases of ES/pPNET and 15 cases of other small round cell tumors (including 8 cases of rhabdomyosarcoma, 4 cases of synovial sarcoma, 2 cases of neuroblastoma and 1 case of lymphoma) using formalin-fixed and paraffin-embedded tissues. RESULTS: EWS-FLI1/ERG fusion transcript was detected in 20 of the 25 ES/pPNET cases (80%). The 15 non-ES/pPNET control cases were negative for EWS-FLI1/ERG fusion transcript. CONCLUSIONS: Detection of EWS-FLI1/ERG fusion transcript is a reliable index for molecular diagnosis of ES/pPNET. One-step RT-PCR is a practical method for such analysis in routine paraffin-embedded tumor tissues.

Adolescent↗

PubMed1991

RNA extracted from paraffin-embedded human tissues is amenable to analysis by PCR amplification.

Fixed and paraffin-embedded tissues from pathology department archives can be available for RNA expression analysis. In this report, we show that RNA isolated from surgical or autopsy tissues, routinely processed by fixation and paraffin embedding, is not completely degraded. RNA fragments around 100-200 bases in length are still present even in organs late fixed and very rich in RNase, such as the pancreas. Here we describe a general protocol to obtain RNA from single 6-8-microns tissue sections. The RNA extracted can be analyzed for the presence of specific sequences by reverse transcription and amplification with the PCR. We studied the retinoblastoma gene expression in 38 human pancreas specimens from surgical or autopsy origin.

Animals↗

PubMed1997

Multi-parameter flow cytometric analysis with detection of the Ki67-Ag in paraffin embedded mammary carcinomas.

In the present study we describe a novel multiparameter flow cytometric (FCM) assay to estimate the fraction of cycling cells in epithelial tumors derived from fresh frozen as well as archival material. To this end, MCF-7 cells as well as a series of breast carcinomas (n = 10; fresh frozen as well as formalin fixed and paraffin embedded) were stained using a panel of different antibodies directed against the Ki67-Ag (DAKO/PC, MIB-1, Ki-S5, and poly-Ki67) for a 3-parameter cytokeratin/Ki67-Ag/DNA FCM analysis. Whereas all Ki67-Ag antibodies work equally well in the methanol fixed cell line, MIB-1 and Ki-S5 epitopes are retained in cell suspensions mechanically derived from fresh frozen tissue. Only antibody Ki-S5 shows specific nucleolar staining patterns in cell suspensions prepared by trypsin digestion of formalin fixed, paraffin embedded tissue sections. A good correlation was found between the fractions of Ki67-Ag-positive epithelial cells measured in cell suspensions derived from fresh frozen and the corresponding formalin fixed and paraffin embedded tumor samples. Furthermore, the fraction of Ki67-Ag-positive epithelial cells as determined by 3-parameter FCM correlated very well with the Ki67-Ag-labeling index in paraffin embedded tissue sections.

Antibodies, Neoplasm↗

PubMed1988

Immunohistochemical characterization of two new monoclonal antibodies (LN-4, LN-5) reactive with human macrophage subsets and derived malignancies in B5-fixed, paraffin-embedded tissues.

Two monoclonal antibodies (LN-4, LN-5) reactive to human macrophages in B5 formalin-fixed, paraffin-embedded tissue sections have been produced by using deparaffinized cell extracts of peripheral blood mononuclear cells. Both monoclonal antibodies were initially identified on paraffin-embedded sections of hyperplastic lymph nodes by using the immunoperoxidase staining procedure. Specificity screens on normal human tissues show that LN-4 and LN-5 stain the cytoplasm of macrophages and histiocytes in hematopoietic organs including Kupffer's cells of the liver and Langerhans' cells of the skin. LN-4 also showed strong positivity with acini of the stomach, whereas LN-5 was positive with mantle zone B lymphocytes of the lymph node and spleen, spermatogonia, and chief cells of the stomach. Both antibodies were strongly reactive with cases of true histiocytic lymphoma but, except for infiltrating macrophages, were entirely negative in Hodgkin's disease and non-Hodgkin's lymphomas. In all cases of nodular sclerosis Hodgkin's disease, LN-4 was positive in macrophagelike cells present in the collagen bands surrounding the Hodgkin's lesions. Both monoclonal antibodies were also positive in macrophages and histiocytes present in a variety of benign lymphoid lesions including persistent generalized lymphadenopathy, Gaucher's disease, sinus histiocytosis, and dermatopathic lymphadenopathy. Because of their specificity for human macrophages, and their ability to stain B5-fixed, paraffin-embedded tissues, LN-4 and LN-5 are important new reagents for the diagnosis and classification of malignant and benign histiocytic lesions.

Animals↗

PubMed1992

Immunohistochemical study of p53 expression in microwave-fixed, paraffin-embedded sections of colorectal carcinoma and adenoma.

Expression of p53, a tumor-suppressor gene product, was studied immunohistochemically in microwave-fixed, paraffin-embedded sections of 84 colorectal carcinomas and 44 adenomas. Using a monoclonal antibody (PAb1801), nuclear p53 was detected successfully in 51 of 84 (60.7%) cases of carcinomas, and no stain for p53 was demonstrated in the adjacent normal mucosa. The results in the microwave-fixed, paraffin-embedded sections correlated with those in the frozen sections. The incidence of p53 expression in colorectal carcinomas was high in the cases with distant metastasis, but it was not affected by clinicopathologic features such as tumor size or depth of invasion. In colorectal adenomas, only 4 of 44 (9%) adenomas expressed p53. This expression of p53, however, was restricted to only a few glands within tubular adenomas with mild dysplasia. Thus, p53 protein was expressed preferably by malignant tumors of the colorectum. The present study demonstrated the usefulness of microwave fixation to preserve p53. The immunohistochemical detection of p53 in microwave-fixed, paraffin-embedded sections of colorectal carcinoma and adenoma can provide valuable information about the mechanism of carcinogenesis in colorectal epithelium.

Adenoma↗

PubMed1997

Prognostic significance of Ki-67 antigen and p53 protein expression in pancreatic duct carcinoma: a study of the monoclonal antibodies MIB-1 and DO-7 in formalin-fixed paraffin-embedded tumour material.

Formalin-fixed paraffin-embedded material from 57 patients in whom curative resection of pancreatic carcinoma had been attempted was analysed by an immunohistochemical procedure to estimate proliferation and p53 protein expression. Using the monoclonal antibody (MAb) MIB-1, which recognizes a Ki-67 epitope, the proliferating cell index (PCI, percentage of immunoreactive tumour nuclei) and proliferating cell area (PCA, percentage of immunoreactive tumour nuclear area) were calculated using an interactive image analysis system and were compared with semiquantitative scoring of stainability. MAb DO-7, which recognizes both wild- and mutant-type p53 protein, was used to assess p53 expression in the same material. MIB-1 stainings were of high quality in 53 tumours. The median PCI was 29.7% (range 0.5-82.1%) and the median PCA was 10.6% (range 0.0-36.5%). There was a close correlation between PCI and PCA (P < 0.0001). PCI and PCA values were in conformity with the semiquantitative scoring (P < 0.0001). The p53 immunohistochemical stainings were successful in 48 tumours and the protein was expressed in 22 (46%). High PCI values (> 45%, n = 14) correlated with shorter survival time (P < 0.01). PCA (P < 0.05) and the expression of p53 protein (P < 0.001) were independent prognostic variables.

Aged↗

PubMed2005

MALDI-TOF MS and TaqMan assisted SNP genotyping of DNA isolated from formalin-fixed and paraffin-embedded tissues (FFPET).

Formalin-fixed paraffin-embedded tissues (FFPET) from archived clinical samples provide an invaluable source for large-scale molecular genetic studies. Pharmacogenetic investigations that require long-term clinical follow-up data of patients may particularly benefit from FFPET analysis. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and TaqMan-based (Thermus aquaticus polymerase) methodologies have become standard genotyping procedures. However, no data are available on the applicability of MALDI-TOF MS to the genotyping of low quality DNA, as it is usually obtained from FFPET, and data from TaqMan genotyping are limited. We isolated constitutional DNA from 274 FFPET samples (229 patients with breast cancer and 45 patients with benign breast diseases) and genotyped 15 polymorphic loci in 10 genes. Nine SNPs were genotyped by MALDI-TOF MS, and six were genotyped by the TaqMan methodology. We established rates for successful allele assignment for all FFPET, for FFPET prepared prior to 1990, and for FFPET prepared post-1990. Both methodologies showed high success rates ranging between 70.9 and 99.6% (mean: 91.8%) for MALDI-TOF MS and between 82.3 and 97.7% (mean: 91.0%) for TaqMan genotyping. No significant differences in genotyping performances for FFPET prepared prior to 1990 or post-1990 were observed. With the exception of one, all other genotype frequencies were in Hardy-Weinberg equilibrium. Furthermore, genotype frequencies matched those observed in a German breast cancer population and other Caucasian populations. Our study shows for the first time that MALDI-TOF MS and TaqMan genotyping procedures provide reliable data, and are therefore applicable in studies that require large scale FFPET genotyping.

Alleles↗

PubMed2006

Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues.

BACKGROUND: Formalin-fixed paraffin-embedded (FFPE) tissues represent the largest source of archival biological material available for genomic studies of human cancer. Therefore, it is desirable to develop methods that enable whole genome amplification (WGA) using DNA extracted from FFPE tissues. Multiple-strand Displacement Amplification (MDA) is an isothermal method for WGA that uses the large fragment of Bst DNA polymerase. To date, MDA has been feasible only for genomic DNA isolated from fresh or snap-frozen tissue, and yields a representational distortion of less than threefold. RESULTS: We amplified genomic DNA of five FFPE samples of normal human lung tissue with the large fragment of Bst DNA polymerase. Using quantitative PCR, the copy number of 7 genes was evaluated in both amplified and original DNA samples. Four neuroblastoma xenograft samples derived from cell lines with known N-myc gene copy number were also evaluated, as were 7 samples of non-small cell lung cancer (NSCLC) tumors with known Skp2 gene amplification. In addition, we compared the array comparative genomic hybridization (CGH)-based genome profiles of two NSCLC samples before and after Bst MDA. A median 990-fold amplification of DNA was achieved. The DNA amplification products had a very high molecular weight (> 23 Kb). When the gene content of the amplified samples was compared to that of the original samples, the representational distortion was limited to threefold. Array CGH genome profiles of amplified and non-amplified FFPE DNA were similar. CONCLUSION: Large fragment Bst DNA polymerase is suitable for WGA of DNA extracted from FFPE tissues, with an expected maximal representational distortion of threefold. Amplified DNA may be used for the detection of gene copy number changes by quantitative realtime PCR and genome profiling by array CGH.

Carcinoma, Non-Small-Cell Lung↗

PubMed2002

Toward efficient analysis of mutations in single cells from ethanol-fixed, paraffin-embedded, and immunohistochemically stained tissues.

Only a few studies have demonstrated successful molecular analysis after whole genome amplification using single cells dissected from paraffin-embedded tissues. The results in these studies were limited by low-amplification efficiency and high rates of allele dropout. In the present study, the amplification rate using a thoroughly modified primer extension and preamplification-PCR protocol was improved significantly for single cells microdissected from paraffin-embedded and immunohistochemically stained tissues. Tissue fixation with ethanol (85%) and the addition of 0.2 mmol/L EDTA helped to achieve an amplification rate between 67% (segments 200 to 400 bp) and 72% (segments <200 bp). Normal tissue sections were immunohistochemically double stained for overabundance of p53 protein and proliferating cell nuclear antigen. Microdissection of single cells was performed with a manual micromanipulator equipped with a Tungsten needle. Sequence analysis of the TP53 gene was performed after improved primer extension preamplification-PCR and multiplex PCR from single microdissected cells. The rate of allele dropout was at least 68%. These technical advances facilitate routine mutation analysis using a single cell or a few cells microdissected from routinely processed paraffin-embedded normal and tumor tissues. Allele dropout still represents a serious problem in single-cell mutation analysis, especially in samples with limited template DNA and prone to DNA damage.

Breast↗

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↗

PubMed1995

A cautionary note regarding the application of Ki-67 antibodies to paraffin-embedded breast cancers.

Immunocytochemical studies examining the Ki-67 proliferation marker in paraffin-embedded material have recently been made possible by the availability of several antibodies, notably MIB-1, which are readily applicable to microwaved sections. Using breast cancer material, the present study examines correlations shown by these new paraffin assays and also by PCNA (proliferating cell nuclear antigen), an existing marker of proliferation, with the established Ki-67 cryosection assay. Paraffin sections were microwaved prior to incubation with Ki-67 or MIB-1 antibodies. Signal detection was carried out with a biotinylated secondary antibody, peroxidase-conjugated streptavidin, and DAB/H2O2 chromogen. The results suggest that caution is required when studying proliferation in paraffin-embedded breast cancers by immunostaining using Ki-67 antibodies. Nuclear staining in wax sections (Ki-Par, MIB-1, PCNA) greatly exceeded that in cryosections (Ki-Froz) and thus correlations were notably absent between Ki-Par or PCNA immunostaining and the routine Ki-Froz assay. Immunostaining with MIB-1 or PCNA may, however, be useful to assess proliferation if cut-offs are applied to eliminate weak immunostaining associated with wax sections. Thus, an approximately linear relationship was seen between MIB-1/Ki-Froz, which was improved if only moderately or moderately/strongly MIB-1-positive cells were scored. Similarly, a significant correlation was also revealed between PCNA/Ki-Froz if such a cut-off was applied.

Antibodies, Monoclonal↗