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Results for “Paraffin Embedding”

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PubMed1988

Purification and analysis of RNA from paraffin-embedded tissues.

We have developed methods for the purification and analysis of RNA from formalin-fixed and paraffin-embedded tissue. The methods allow retrospective analysis of gene expression or viral infection. RNA extraction uses prolonged solubilization of tissue with detergent and protease in the presence of high concentrations of a ribonuclease inhibitor. The purified RNA is moderately degraded but its hybridization behavior is essentially unaffected. We were able to quantify specific mRNAs by dot-blot hybridization.

Animals↗

PubMed2005

Development of new rabbit monoclonal antibody to estrogen receptor: immunohistochemical assessment on formalin-fixed, paraffin-embedded tissue sections.

Evaluation of estrogen and progesterone receptors in breast cancer is widely used for the prediction of the response to endocrine therapy and as a biologic parameter closely related to disease prognosis. Immunohistochemistry is considered a specific, sensitive, and economic method for the determination of estrogen receptor/progesterone receptor status. The authors developed the first rabbit antiestrogen receptor monoclonal antibody (clone SP1) used in immunohistochemistry on formalin-fixed, paraffin-embedded tissue sections especially from breast carcinomas. This new antibody, compared with currently available antiestrogen receptor antibodies, has important advantages, including its reactivity even without heat-based antigen retrieval of fixed, embedded tissue sections in immunohistochemistry, and the predominance of nuclear immunostaining with only a very low cytoplasmic signal. A comparative study of immunohistochemistry on 61 histologic specimens from breast cancer cases showed that SP1 yields the same results as the well-known, standardized mouse monoclonal antibody to estrogen receptor (clone 1D5). Antibody affinity of SP1 is 8 times higher than that of 1D5. Thus, SP1 may prove of great value in the assessment of estrogen receptor status in human breast cancer.

Animals↗

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↗

PubMed1980

Cytophotometric DNA measurements of chondrosarcoma: methodologic aspects of measurements in tissue sections from old paraffin-embedded specimens.

The methodologic prerequisites of cytophotometric DNA measurements of normal and tumor cells in tissue sections obtained from paraffin blocks and preserved as archival material were investigated. The optimal time of hydrolysis in 5-N HCl at room temperature was one hour for the different cell types analyzed. Paraffin-embedded tissues, stored for two decades, were still suitable for quantitative cytophotometric DNA determinations of Feulgen-stained nuclei. Different cell types in the Feulgen-stained sections could be identified with accuracy. The 90th percentile of fibroblast (internal control cells) DNA values was used as an upper limit of the diploid DNA content. By determining the number of tumor cells with DNA values exceeding this limit, nondiploid (hyperploid) tumor cell populations could be discriminated from diploid tumor cell populations. Cell population analysis of ploidy level, performed in this way, was found to be accurate in tissue sections of 4 micrometer. The accuracy of this analysis was not improved by increasing the section thickness. Since tissue sections obtained from old paraffin blocks could be used for the determination of hyperploidy, the prognostic significance of this parameter in different tumors can be assessed retrospectively.

Cell Nucleus↗

PubMed2003

In situ detection of O6-methylguanine-DNA methyltransferase messenger RNA in paraffin-embedded human astrocytic tumor tissues by nested in situ RT-PCR is useful in predicting chemotherapy-resistance of tumors.

O6-methylguanine-DNA methyltransferase (MGMT) is an enzyme that interferes with chemotherapeutic effect of alkylating agents. We performed in situ detection of MGMT mRNA utilizing the nested RT-PCR method in tissue sections (nested in situ RT-PCR). We analyzed 34 samples of paraffin-embedded astrocytic tumor tissue sections with this method [3 astrocytomas, 14 anaplastic astrocytomas (AA), and 17 glioblastoma multiformes (GBM)]. Twenty-five cases (73.5% of all cases) were positive for MGMT either with our method or immunohistochemistry (IHC). Moreover, with our method >25% of the cells in the tumor tissue expressed MGMT in contrast to >4% with IHC among the population of MGMT positive cases. Our method was significantly more sensitive than IHC (p=0.0004). The present results suggest that potentially there is a greater population of MGMT positive cells in astrocytic tumor tissues than the one evaluated with IHC. These findings suggest that the >25% of the MGMT positive cells are involved in the interference with the chemotherapeutic effect of alkylating agents. The MGMT expressing cell population was markedly decreased in GBM compared with AA (26.1% vs 62.1%). The main reason for this marked decrease was that MGMT was expressed in only 9 of 17 cases of GBM in contrast to all AA cases that expressed MGMT. This result suggests that there are potentially two populations of GBM on the basis of MGMT expression, in which the negative population might be mainly composed of de novo GBM. Therefore, it is suggested that our method is practically useful to detect any drug resistance gene product with high sensitivity and would provide a chance to evaluate the chemotherapeutic effect of any agents in an individual patient based manner.

Astrocytoma↗

PubMed2006

Automated analysis of fluorescence in situ hybridization on fixed, paraffin-embedded whole tissue sections in B-cell lymphoma.

Certain recurrent cytogenetic abnormalities are diagnostic of a specific neoplasm and may portend prognosis. As conventional cytogenetics may not reveal a neoplastic clone, and unfixed material for fluorescence in situ hybridization may be unavailable, performing fluorescence in situ hybridization on fixed tissues is diagnostically and prognostically valuable. Manual interpretation of fluorescence in situ hybridization signals may be difficult on paraffin-embedded tissue sections due to truncated nuclei. Therefore, we investigated the use of an automated image acquisition and analysis system (MetaSystems) for interpretation of fluorescence in situ hybridization signals in tissue sections from dual fusion translocation probes. Three probe sets were analyzed on archival specimens with a confirmed diagnosis of mantle cell lymphoma, follicular lymphoma or Burkitt lymphoma. 100% of mantle cell lymphomas (7/7) were positive for t(11;14), 91% of follicular lymphomas (10/11) for t(14;18) and 100% of Burkitt lymphomas (9/9) for t(8;14). Successful hybridization was achieved using various tissue fixatives and fluorescence in situ hybridization interpretation was blinded with respect to the underlying diagnosis. Based on these results, automated analysis of fluorescence in situ hybridization on fixed tissues is accurate and valuable in the evaluation of B-cell lymphoma, and may provide pertinent diagnostic and prognostic information.

Burkitt Lymphoma↗

PubMed1984

Localisation of enteropathogens in paraffin embedded tissue by immunoperoxidase.

An indirect immunoperoxidase technique has been used to identify enteropathogens in formol-sublimate fixed paraffin embedded sections of calf intestine. Infections with bovine rotavirus, bovine coronavirus, Newbury agent SRV -1, and K99+ Escherichia coli have been detected in the intestines from experimentally infected and conventially reared diarrhoeic or normal calves. The ability to visualize enteropathogenic agents in histological sections resulted in the demonstration of virus infected cells at sites not previously shown to be infected using the immunofluorescence technique.

Animals↗

PubMed2001

Polymerase chain reaction in the diagnosis of T-cell lymphoma in paraffin-embedded bone marrow biopsies: a comparative study.

AIMS: In routine histological analysis of bone marrow biopsies, the distinction between reactive T-cell infiltrates and T-cell lymphoma can be difficult, even with the use of extensive immunohistochemistry. The aim of this study was to evaluate the diagnostic contribution of TCR-gamma gene rearrangement analysed by PCR. METHODS AND RESULTS: The samples studied consisted of 46 paraffin-embedded bone marrow biopsies (diagnosis, staging and follow-up) from 26 patients with T-cell lymphoma. The bone marrow biopsies were categorized into three groups according to the morphological and immunohistochemical results. Group 1, positive for T-cell lymphoma (24 bone marrow biopsies), group 2, suspicion of T-cell lymphoma (15 bone marrow biopsies) and group 3, negative for T-cell lymphoma (seven bone marrow biopsies). DNA could be amplified in 45/46 bone marrow biopsies (98%). Clonal rearrangement was detected in 30/45 bone marrow biopsies tested (67%) including 15/24 bone marrow biopsies (62.5%) of group 1, 11/14 (78.5%) of group 2 and 4/7 (57%) of group 3. In total, PCR analysis supported a diagnosis of T-cell lymphoma in 15/45 bone marrow biopsies (33%), in which histological and/or immunohistochemical examination provided inconclusive evidence of malignancy. CONCLUSIONS: TCR-gamma PCR is a complementary tool for the assessment of T-cell lymphoma in bone marrow biopsies. Optimal evaluation of bone marrow biopsies requires an integrative approach of all available results from morphology, immunohistochemistry, molecular biology and clinical data.

Biopsy↗

PubMed1994

Flow cytometric DNA content analysis of paraffin-embedded tissue derived from cervical carcinoma.

PURPOSE: Flow cytometric deoxyribonucleic acid (DNA) content analysis has been shown to be of prognostic importance in some cancers. There have been recent reports of a prognostic importance for DNA content analysis in cervical carcinoma. METHODS AND MATERIALS: We retrospectively reviewed the hospital and radiation oncology records of cervical carcinoma patients who presented between 1984-1990. RESULTS: A total of 101 archival paraffin-embedded blocks were processed, of which 77 were of technical quality for analysis. Thirty-five percent were found to be DNA content aneuploid (DNA-A) and 65% DNA content diploid (DNA-D). No statistical difference was found between the two groups in age at diagnosis, % S-phase, coefficient of variation (CV), or proliferative index (PI). A statistical difference was noted in the G2M phase between the two groups (p = 0.004). The median % S-phase was 8.4% in the DNA-D group. A statistical difference (p = 0.017) in survival was noted between the low and high % S-phase DNA-D groups. In patients who received radiation alone, high-PI patients had improved survival compared to low-PI patients. No statistical difference in survival was noted in the high % S-phase DNA-D group and DNA-A group (p = 0.28). Proportional Hazard (Cox) Regression found clinical stage the only independent prognostic indicator for survival. CONCLUSION: Flow cytometric DNA content analysis is being used more frequently in the management of different malignant tumors. Our study shows that DNA content analysis is useful in determining the prognosis and survival outcomes in cervical carcinomas and may aid in predicting outcome to certain types of treatment regimens.

Aneuploidy↗

PubMed1998

Comparison of histologic stains for use in PCR analysis of microdissected, paraffin-embedded tissues.

We evaluated the effect of six different histologic stains on the productivity of PCR amplification of DNA isolated from paraffin-embedded tissue samples. The tissue was collected from glass slides by microdissection techniques, whereby tiny portions of tissue are visually identified through a microscope and selectively resected for subsequent DNA extraction and PCR amplification. We found that the success of PCR amplification depended on the type of histologic stain that was used to facilitate microscopic visualization of the undeparaffinized tissue section. The best results were obtained with methyl green and nuclear fast red, while Wright's stain yielded less PCR product. Two other stains, Evans blue and light-green SF yellowish (also known as the counterstain for geomori methenamine silver stain) yielded sufficient PCR products; however, their staining characteristics did not afford satisfactory visualization of nuclear chromatin to discriminate between benign and malignant cells. Our most significant finding was that a commonly used histologic stain, hematoxylin, failed to produce DNA templates that could be consistently amplified by PCR. In conclusion, it is prudent to avoid hematoxylin stains when preparing tissues as starting material for PCR. Among the remaining five stains that were evaluated, the best choice depends on the differential staining characteristics of the cells to be dissected.

Coloring Agents↗

PubMed2005

Immunohistochemical analysis of a muscle ankyrin-repeat protein, Arpp, in paraffin-embedded tumors: evaluation of Arpp as a tumor marker for rhabdomyosarcoma.

Arpp, a protein including an ankyrin-repeat, P EST motif, and p roline-rich region, is a recently identified protein that is exclusively expressed in striated muscles. This study comprehensively analyzed its expression among soft tissue sarcomas of various histological types and evaluated its potential use for the differential diagnosis of rhabdomyosarcoma (RMS). Formalin-fixed, paraffin-embedded tissues, including 37 RMS cases, 88 non-RMS sarcomas, and 38 carcinomas, were analyzed for Arpp expression. Arpp was detected in 33 (89.2%) of 37 RMS cases by immunohistochemistry. Western blot analysis revealed expression of Arpp in all RMS cases tested. High expression of Arpp was generally associated with morphological evidence of skeletal muscle differentiation of tumor cells. In contrast, Arpp displayed 6.3% (8/126) positivity among the non-RMS tumors. Focal or weak expression was seen in malignant fibrous histiocytoma (2/27), synovial sarcoma (1/11), Ewing sarcoma (1/5), and epithelioid sarcoma (3/5), whereas one epithelioid sarcoma displayed strong expression for Arpp. A comparative analysis of the Arpp profile with that of myogenic markers in RMS revealed that the sensitivity of Arpp (89.2%) was higher than that of myoglobin (59.6%) and comparable with that of myogenin (88.2%), MyoD (80.6%), muscle-specific actin (83.8%), and desmin (89.2%). These results suggested that Arpp is sensitive to and specific for RMS. Thus, we proposed that Arpp is a novel skeletal muscle-specific marker, which is useful for differential diagnosis of RMS.

Biomarkers, Tumor↗

PubMed1999

Detection of t(2;5) in anaplastic large cell lymphoma: comparison of immunohistochemical studies, FISH, and RT-PCR in paraffin-embedded tissue.

Anaplastic large cell lymphoma (ALCL) is associated with the t(2;5)(p23;q35) translocation involving the anaplastic lymphoma kinase gene (ALK) and the nucleophosmin gene (NPM), which result in expression of a novel fusion protein, NPM-ALK (p80). Clinicopathologic studies have shown that ALK expression in ALCL is associated with improved 5-year survival rates when compared with ALCL lacking ALK expression. This study used paraffin-embedded tissue to compare interphase fluorescence in situ hybridization (FISH) and reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of t(2;5) with immunohistochemical analysis for the detection of ALK protein expression in 27 patients with CD30-positive ALCLs. ALK protein expression was detected with ALK1 antibody in 14 of the 27 patients. The neoplastic cells in 13 of these 14 lymphomas reacted with the p80NPM/ALK antibody. FISH, using a two-color ALK DNA probe, correlated 100% with the immunohistochemical results: a translocation involving the ALK gene was detected in all 14 lymphomas that reacted with anti-ALK1. RT-PCR, performed on 21 lymphomas, detected NPM-ALK mRNA in five of the lymphomas, all of which reacted with anti-ALK1 and showed ALK gene rearrangement by FISH. Lymphomas showing ALK1 reactivity occurred in a younger patient population (median age, 19.5 years) and were associated with improved 5-year survival rates (84%), as compared with lymphomas lacking ALK1 reactivity (median age, 68.0 years; 5-year survival rate, 35%; p = 0.008). We conclude that immunohistochemical studies, using antibody ALK1. and FISH for ALK gene rearrangement are equally effective for identifying patients with ALCL who have a favorable clinical outcome.

Adolescent↗

PubMed2005

Detection of fusion genes in sarcomas using paraffin-embedded tissues.

Many sarcomas are characterized by specific recurrent chromosomal translocations resulting in gene fusions. The genes involved in almost all of these translocations have been cloned, greatly changing sarcoma diagnosis. At the biological level, these chromosomal translocations produce highly specific fusion genes that encode key molecules for tumor development. The clinical correlation between these translocation-derived genetic markers and discrete histopathological entities has been remarkable. Today, detection of fusion genes plays a crucial role in the diagnosis of sarcomas that harbor atypical clinical or pathological presentations. The focus of this brief review is the recent impact that cytogenetic and molecular detection of these translocations has had on sarcoma diagnosis using paraffin-embedded sections.

Animals↗

PubMed2002

Detection of three common translocation breakpoints in non-Hodgkin's lymphomas by fluorescence in situ hybridization on routine paraffin-embedded tissue sections.

Non-random chromosomal translocations are specifically involved in the pathogenesis of many non-Hodgkin's lymphomas and have clinical implications as diagnostic and/or prognostic markers. Their detection is often impaired by technical problems, including the distribution of the breakpoints over large genomic areas. This study reports a fluorescence in situ hybridization (FISH) method which allows the detection of specific chromosomal breakpoints in tissue sections from routinely fixed, paraffin-embedded samples. Hybridization was performed after demasking the DNA. Previously validated locus-specific probes (cosmids, PACs) flanking the BCL1, BCL2 regions and similar new probes for the MYC breakpoint region were used. The cases studied were five mantle cell lymphomas (MCL) and five follicular lymphomas (FL), selected on the basis of a previously proved t(11;14) and t(14;18) and five randomly chosen Burkitt's lymphomas (BL), as well as 21 negative control samples. In all samples, hybridization signals of sufficient intensity were obtained. Three different algorithms were used to score the hybridization signals in tissue sections, two of them taking into account the nuclei and their signal distribution indicative of chromosomal break, and one only considering the colocalization or segregation of the signals. In control tissues, these algorithms resulted in cut-off levels of 9.1%, 1.3%, or 10.0%. In the 15 lymphoma samples the percentages of abnormal cells/signals ranged from 28% to 80%, 13% to 49%, and 40% to 70%, respectively. The results indicate that small locus-specific probes can be used in FISH for regular detection of translocation breakpoints on routine paraffin tissue sections.

Algorithms↗

PubMed1991

Application of lectin and B-lymphocyte-specific monoclonal antibodies for the demonstration of human microglia in formalin-fixed, paraffin-embedded brain tissue.

To evaluate the usefulness of microglial markers for routine neuropathological material, we studied formalin-fixed, paraffin-embedded human brain tissue with the immunoperoxidase method using the lectin Ricinus communis agglutinin (RCA-1) and four monoclonal antibodies (LN-1, LN-2, LN-3, anti-HLA-DR/alpha). RCA-1 stained resting microglia, but the staining intensity was mostly weak. LN-1 also stained resting microglia in paraffin sections first treated with protease. In contrast to LN-1, RCA-1 stained blood vessels heavily. LN-1 stained resting microglia more markedly than RCA-1 in brains fixed for a prolonged period of time. However, LN-1 recognized a small number of astrocytes in routine paraffin sections. LN-3 reactivity was detected on a few resting microglia, but was intensely expressed on large numbers of reactive microglia in many neurological diseases. Both LN-2 and anti-HLA-DR/alpha labelled microglia, but the reactions were inconsistent. This study suggests that the monoclonal antibodies LN-1 and LN-3 are useful for the demonstration of microglia in paraffin sections, and a combination of these antibodies and the antibody to glial fibrillary acidic protein is recommended in attempting to identify microglia.

Antibodies, Monoclonal↗

PubMed2005

Paraffin-embedded manipulated blastocysts: a tool to demonstrate stem cell plasticity?

One of the big question marks in current stem cell research is whether there is true plasticity of adult progenitor cells (APC) or if cell fusion is the principle source of the supposed plasticity. The generation of chimeras by injecting adult progenitor cells into blastocysts is not new. This paper describes an efficient embedding technique for murine blastocysts injected with human APC. This method could help in establishing a novel tool to analyse the process of plasticity, if it truly exists. If this is the case, this technology could be of great help to characterize surface markers of stem cells in great detail. On the other hand, fusion of cells could also be investigated. A system of embedding blastocysts was set up using paraffin for further analysis by means of light microscopy and immunohistochemistry. The embedding of the chimaeras consists of fixing them first with paraformaldehyde in phosphate-buffered saline (PFA/PBS), embedding them in gelatine, fixing the gelatine block with PFA/PBS and finally fixing the gelatine block in a Petri dish by embedding it in paraffin. Using this protocol, the morphology of the blastocysts is well preserved.

Animals↗

PubMed1986

Effect of section thickness on quality of flow cytometric DNA content determinations in paraffin-embedded tissues.

DNA content determinations were carried out by flow cytometry on nuclear suspensions prepared from the same paraffin-embedded tissue block for each of eight surgically resected human carcinomas at section thicknesses of 5,10,20,30,40,50, and 100 millimicrons. Flow cytometric DNA determinations were also obtained on fresh tissue specimens in four of the eight carcinomas. As section thickness decreased below 50 millimicrons, there was a progressive increase in the histogram baseline noise at low DNA values and a decrease in the relative peak height of aneuploid DNA. The former was attributed to an increase of nuclear fragments in thinner sections, and the latter to the greater probability of transection of the larger aneuploid cells within a specimen. Both artifacts were minimized at section thickness of 50 millimicrons or greater.

Breast Neoplasms↗

PubMed1995

Monoclonality in gastric lymphoma detected in formalin-fixed, paraffin-embedded endoscopic biopsy specimens using immunohistochemistry, in situ hybridization, and polymerase chain reaction.

Diagnosis of gastric malignant lymphoma remains a challenge, especially when the tissue source is endoscopic biopsy specimens. Once an atypical lymphoid infiltrate is found, demonstration of clonality is the key to establishing a diagnosis of the disease. For this purpose, we evaluated the usefulness of immunohistochemistry, in situ hybridization, and polymerase chain reaction (PCR) using formalin-fixed, paraffin-embedded endoscopic materials from 20 patients with B-cell malignant lymphomas. Template DNA for PCR was obtained by microdissecting Giemsa-stained sections using a serial hematoxylin and eosin section as a guide. Clonal rearrangement bands were demonstrated in 15 of 20 cases (75%) by PCR, whereas expression of monotypic light-chain mRNA was detected in seven of 20 (35%) by in situ hybridization and monotypic light-chain restriction in four of 20 (20%) by conventional immunohistochemistry. Although less sensitive than PCR, in situ hybridization was useful for localizing the expression of target mRNAs with cellular accuracy and with low background staining. In addition, two cases were found to be monoclonal only by in situ hybridization, and not by PCR. The results showed that clonal proliferation is detected with the greatest sensitivity with PCR using small routinely processed biopsy specimens and that a difficulty with the PCR method in terms of cellular localization was partially overcome using a microdissection procedure that provided at least tissue-level accuracy.

Base Sequence↗