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PubMed1991

Feasibility of in situ hybridisation with chromosome specific DNA probes on paraffin wax embedded tissue.

The feasibility was studied of in situ hybridisation using chromosome specific DNA probes on paraffin wax embedded normal and malignant tissues from different organs. Both isolated nuclei and 5 microns sections were used in in situ hybridisation experiments with biotinylated repetitive DNA probes specific for the centromeric regions of chromosomes 1 and 17. The hybridisation results were visualised with peroxidase-diaminobenzidine. The optimal pretreatments with sodium thiocyanate and pepsin were experimentally defined for the different tissues. Although interphase cytogenetics on paraffin wax embedded tissue is possible, the results indicate that it has its limitations, compared with investigations on fresh tumour tissue.

Chromosomes, Human, Pair 1↗
PubMed1996

Immunohistochemical detection of Ki-67 in epithelial skin tumors in formalin-fixed paraffin-embedded tissue sections using a new monoclonal antibody (MIB-1).

The expression of the Ki-67 antigen was investigated in 44 epithelial skin tumors using an immunohistochemical technique on formalin-fixed, paraffin-embedded tissue sections. Microwave oven heating was employed for retrieval of the antigen in these tissue sections. The staining patterns varied among the epithelial skin tumors. The assessment of immunohistochemical staining was based upon the growth fraction (GF), defined as the number of Ki-67 positive cells divided by the total number of tumor cells counted and expressed as a percentage. GF was 9.7 +/- 3.1% in seborrheic keratosis, 19.5 +/- 2.9% in keratoacanthoma, 23.1 +/- 4.9% in basal cell carcinoma, 18.5 +/- 6.3% in actinic keratosis, 37.1 +/- 6.0% in Bowen's disease, and 32.9 +/- 10.5% in squamous cell carcinoma. There was a significant difference in GF between the keratoacanthoma and squamous cell carcinoma (p < 0.01). Actinic keratosis showed a relatively low GF, whereas Bowen's disease showed a high one. Furthermore, the GF tended to increase with tumor cell differentiation in squamous cell carcinoma: 23.7% (+/- 5.0) in well-differentiated, 35.0% (+/- 6.2) in moderately-differentiated, and 47.6% (+/- 4.5) in poorly-differentiated squamous cell carcinomas. Immunohistochemistry with MIB-1 may give useful additional information in the differential diagnosis of KA and SCC.

Antibodies, Monoclonal↗
PubMed1989

Immunohistological localisation of rinderpest virus in formalin-fixed, paraffin-embedded tissues from experimentally infected cattle.

Six Freesian steers were subcutaneously inoculated with the virulent rinderpest virus strain Kabete "0" and sacrificed at the height of fever. Sections of formalin-fixed, paraffin-embedded tissues were stained according to the peroxidase anti-peroxidase (PAP) technique. Labelling of viral antigen, both in the cytoplasm and in the nuclei of infected cells, was observed in the epithelial cells of the upper and lower alimentary tract and in lymphoid organs, i.e. spleen, lymph nodes, pharyngeal tonsils, Peyer's patches and thymus. Electron microscopy studies confirmed the results.

Animals↗
PubMedDate not supplied

[Analysis of immunoglobulin heavy chain genes rearrangement by PCR from paraffin-embedded tissue in B-cell lymphomes in Tunisia].

OBJECTIVE: To study the lymphoid clonality on Tunisian B-cell lymphomas cases by polymerase chain reaction (PCR)-based techniques using DNA from paraffin-embedded tissues. MATERIAL AND METHODS: Here we conducted a retrospective PCR clonality study on 73 cases of B-cell lymphomas and 12 reactive lymphoid tissues. The quality of DNA extracted was tested by beta-globin PCR. Consensus primers directed at the FRIII-VH and FRII-VH regions of the immunoglobulin heavy chain (IgH) gene were used to detect clonality. RESULTS: The results showed that 52 of 73 (71%) B-cell lymphomas exhibited good quality of amplifiable DNA. Clonality was found in 77% of cases using the set of primers FRIIIa/LJH/VLJH and in 65.5% using the set of primers FRIIa/LJH/VLJH. Lymphomas derived from pregerminal centre showed a high rate detection of clonal IgH gene rearrangement (100%) compared to other group of tumors derived from germinal centre or postgerminal centre (74.5%). None of the polyclonal controls gave a clonal pattern. CONCLUSION: This is the first large series of PCR clonality study of IgH gene rearrangements on B-cell lymphoma from Tunisia. Our results were similar to other reports in terms of sensitivity and specificity of these techniques and confirm the interest of that PCR for detecting clonal IgH gene rearrangements in lymphoma.

Base Sequence↗
PubMed1993

Discrimination between immunoaccessory and phagocytic monocytes/macrophages of the skin in paraffin-embedded tissue by the monoclonal antibody Ki-M1P.

The variety of functions performed by monocytes and macrophages is reflected in their phenotypic diversity. Investigation of this complex system is facilitated by Ki-M1P, a new monoclonal antibody which recognizes a differentiation antigen on monocytes/macrophages in paraffin-embedded tissues. To test its usefulness as a pan-macrophage marker in the skin, we immunohistochemically analysed paraffin-embedded biopsy material from seven healthy individuals and 190 patients with a variety of dermatoses. Immunoreactivity was compared with results obtained with the antibodies KP-1, MAC-387, UCHL-1 and S-100. In normal skin, epidermal Langerhans cells were Ki-M1P-. Strong expression of this marker was detected on spindle-shaped as well as dendritic perivascular and intervascular macrophages. Pathological reaction forms such as giant cells and epithelioid cells in granulomatous dermatoses were also Ki-M1P+. The high specificity of Ki-M1P is reflected in the lack of reactivity with tumour cells in non-monocytic neoplasms and Langerhans cell histiocytosis. Thus, Ki-M1P is a useful marker for skin macrophages, discriminating between the immunoaccessory and the phagocytic compartments.

Antibodies, Monoclonal↗
PubMed1998

Nested polymerase chain reaction for Mycobacterium tuberculosis IS6110 sequence on formalin-fixed paraffin-embedded tissues with granulomatous diseases for rapid diagnosis of tuberculosis.

We evaluated the sensitivity and specificity of a nested polymerase chain reaction (PCR) to the Mycobacterium tuberculosis IS6110 sequence on formalin-fixed paraffin-embedded tissue samples from patients with tubercular and other granulomatous lesions. Five groups of patients and samples were studied: (1) 28 samples from HIV-positive patients with tuberculosis, (2) 8 samples from HIV-negative patients with histologically suspected tuberculosis (confirmed by culture in 5 cases), (3) lymph nodes from 5 HIV-positive patients with Mycobacterium avium-intracellulare infection, (4) lymph nodes from 30 patients with sarcoidosis, and (5) specimens from 17 patients with other granulomatous diseases. The DNA was extracted from sections with a total thickness of 60 microm, and PCR amplified an internal fragment of 123 base pairs. All of the cases with M. tuberculosis infection were PCR-positive, although this sensitivity was partially related to the initial concentration of the DNA used for amplification. Two of the group 4 samples also were repeatedly positive, thus reducing the specificity of the method. All of the cases with granulomatous diseases other than sarcoidosis were negative. We propose a simplified and highly sensitive nested PCR for the diagnosis of M. tuberculosis infection on archived material in HIV-positive and HIV-negative patients.

AIDS-Related Opportunistic Infections↗
PubMed1991

Immuno- and enzymehistochemical characterization of 'plasmacytoid T-cells' in formalin-fixed paraffin-embedded tissue of reactive lymph nodes.

In eight (10.4%) of 77 randomly selected cases of lymphadenitis, foci of 'plasmacytoid T-cells' (PTCs) were found. We characterized the PTCs in formalin-fixed paraffin-embedded tissue by immuno- and enzymehistochemical techniques. In each case the PTCs formed nodular and patchy infiltration patterns around the high endothelial venules in the paracortex of the lymph nodes. The PTCs in half of the cases were present near the intermediary peritrabecular sinuses. The PTCs were found to be positive for CD 45 (LCA), CD 45 R (4 KB 5) and CD 43 (MT 1), but not for CD 45 RO (UCHL 1), L 26 (CD 20), myeloperoxidase, naphthol AS-D chloroacetate esterase, Mac 387, alpha-1-antichymotrypsin or S 100 protein and anti-dendritic reticulum cell antibody. Some lysozyme+ PTCs were mingled in and around the foci in each case. In five cases the PTCs showed weaker positive reactions for CD 68 (EBM 11) and HLA-DR than for T-zone macrophages and sinus histiocytes. Related to the degree of desintegration of the nodular PTC-foci increasing numbers of T- and B-lymphocytes, T-zone macrophages and interdigitating reticulum cells with HLA-DR were detected in the foci. In two cases, where there was no HLA-DR expression by PTCs, small amounts of HLA-DR in secondary lymph follicles and a decrease of HLA-DR+ T-zone macrophages and interdigitating reticulum cells were observed. Our results suggest that there is a high degree of interaction between PTCs and other cellular components in the lymph node. The PTC may represent a special type of resident macrophage with secretory capacities.

Adult↗
PubMed1990

Induction of a heat shock gene (hsp70) in rabbit retinal ganglion cells detected by in situ hybridization with plastic-embedded tissue.

Elevation of body temperature by 2-3 degrees C induces a 2.7 kilobase hsp70 mRNA species in the rabbit retina within 1 hr. In situ hybridization with thin sections derived from plastic-embedded tissue permitted a higher level of resolution of retinal cell types compared to procedures which involved the use of frozen tissue sections. A prominent induction of hsp70 mRNA in retinal ganglion cells was observed when an hsp70 riboprobe was utilized for in situ hybridization. These results indicate that this neuronal cell type responds rapidly to fever-like body temperatures by inducing one of the major heat shock genes.

Animals↗
PubMed1984

Ruthenium red staining of polyanion containing structures in sections from epoxy-resin embedded tissues.

Staining by ruthenium red (0.5 mg/ml in borate buffer at pH = 9.2) has been used for light and electron microscopic visualization of polyanion containing structures in sections from glutaraldehyde-fixed, epoxy-embedded tissues. This staining technique can be applied in a simple and rapid way, showing the reactive cell components with suitable resolution and contrast. Preliminary spectrophotometric studies show the correspondence in absorption characteristics of the dye which is bound to polyanions in situ or in vitro.

Animals↗
PubMed1992

Monoclonal antibody-based immunohistochemical detection of bovine viral diarrhea virus in formalin-fixed, paraffin-embedded tissues.

Thirty-two monoclonal antibodies directed against epitopes on bovine viral diarrhea virus proteins and glycoproteins were tested for immunohistochemical reactivity with bovine viral diarrhea virus in formalin-fixed and paraffin-embedded tissues from 45 cases of bovine viral diarrhea virus-associated mucosal disease. Only one antibody, designated 15C5, which reacts with the 48-kD glycoprotein of bovine viral diarrhea virus, detected an epitope preserved in these specimens. Monoclonal antibody 15C5 and a polyclonal antibody to bovine viral diarrhea virus successfully detected bovine viral diarrhea viral antigens in 44/45 cases of mucosal disease and did not react with formalin-fixed tissues from 30 uninfected cattle. Monoclonal antibody-based immunohistochemical detection of bovine viral diarrhea virus is routinely fixed tissue specimens has advantages over other currently available techniques in terms of the convenience of specimen submission, the relative ease of method standardization, and the rapidity of the test, and by enabling identification of the virus in association with specific tissues, cell types, and histologic lesions.

Animals↗
PubMed1990

Type-specific human papillomavirus detection in formalin-fixed, paraffin-embedded tissue sections using nonradioactive deoxyribonucleic acid probes.

DNA probes directly conjugated to horseradish peroxidase have been used successfully to detect human papillomavirus (HPV) types 6/11, 16, and 18 in formalin-fixed, paraffin-embedded tissue sections. By using silver enhancement of a heavy metal-modified diaminobenzidine precipitate, the sensitivity of human papillomavirus detection was significantly increased without compromising specificity. In studies comparing the specificity of the horseradish peroxidase-labeled probe/silver enhancement system to that of a biotinylated-DNA probe/streptavidin-alkaline phosphatase system, the former was found to be superior.

3,3'-Diaminobenzidine↗
PubMed1985

Leu-M1--a marker for Reed-Sternberg cells in Hodgkin's disease. An immunoperoxidase study of paraffin-embedded tissues.

Monoclonal antibody to Leu-M1, a granulocyte-related differentiation antigen, represents a highly effective reagent for detection of diagnostic Reed-Sternberg (R-S) cells and variants in paraffin-embedded tissues of Hodgkin's disease. In 69 of 73 cases of Hodgkin's disease (41 nodular sclerosis, 25 mixed cellularity, 4 lymphocyte predominance, and 3 lymphocyte depletion types), R-S cells were strongly immunoreactive for Leu-M1. Four cases of lymphocyte predominance Hodgkin's disease (nodular) were uniformly nonreactive for Leu-M1. In most of the positive cases (57/69, 83%), the majority (60-90%) of R-S cells and variants exhibited immunoreactivity for Leu-M1. A characteristic staining pattern included granular and/or vesicular cytoplasmic immunoreactivity, often with a prominent globular paranuclear reaction product, and membrane staining with highly irregular cytoplasmic borders. Evaluation of B-cell (37 specimens), T-cell (20 specimens), and true histiocytic (3 specimens) neoplasms and a case of mastocytosis revealed immunoreactivity for Leu-M1 only in 1 B-cell and 4 T-cell malignancies. The staining patterns in these cases, however, clearly differed from that observed for R-S cells. Studies of nonneoplastic lymphoid tissues (38 total) demonstrated that lymphoid cells were typically nonreactive; histiocytes revealed variable reactivity for Leu-M1. Occasional histiocytes of the sinusoidal network of lymph nodes, particularly in toxoplasmic lymphadenitis, exhibited a staining pattern (membranous/cytoplasmic/paranuclear) similar to that observed for R-S cells. Leu-M1 represents a potentially helpful diagnostic discriminant in the assessment of Hodgkin's disease and its distinction from non-Hodgkin's lymphomas and other lymphoid proliferations.

Antibodies, Monoclonal↗
PubMed2004

PolyA PCR amplification of cDNA from RNA extracted from formalin-fixed paraffin-embedded tissue.

RNA extraction still relies almost exclusively on the use of fresh or frozen tissue, limiting the number of samples that can be analyzed, and there is a growing need for means of global mRNA analysis of archived formalin-fixed paraffin-embedded tissue (FFPET). Previous reports of RNA extraction and amplification from FFPET are limited and do not enable global cDNA amplification. This study used polyA PCR to generate globally amplified cDNA from RNA extracted from formalin-fixed paraffin-embedded samples. RNA was extracted from nine routinely processed archival FFPET samples (lymph node, nasopharynx, prostate, lung and bone marrow) using an Ambion Paraffin Block RNA Isolation Kit. Global cDNA was generated by polyA RT-PCR and used in GAPDH specific PCR and PCR for CD33, c-myb, and SNF2. PolyA cDNA was reamplified by polyA PCR and the reamplified cDNA also used in GAPDH PCR. RNA was extracted from all nine samples, but was degraded. PolyA RT-PCR generated cDNA from all samples and was positive for GAPDH PCR in seven. PCR for CD33, c-myb, and SNF2 was positive in all samples tested. Following reamplification, the polyA cDNA remained positive for GAPDH by PCR. The results demonstrate the feasibility of globally amplifying RNA isolated from archival FFPET samples using polyA RT-PCR, which generates a renewable cDNA pool that can be probed for any cDNA species and reamplified as necessary.

Adenosine↗
PubMed1989

Cellular localization of heat shock gene expression in rabbit cerebellum by in situ hybridization with plastic-embedded tissue.

The major cell types in rabbit cerebellum which engage in the expression of a heat shock gene (hsp70) after hyperthermia were identified. This required in situ hybridization on thin sections derived from plastic-embedded tissue. All classes of cerebellar neurons which were examined (Purkinje, granule, and stellate cells) responded by induction of hsp70 mRNA within 1 hr after hyperthermia. Prominent induction of hsp70 mRNA was also observed in oligodendroglia in the deep white matter.

Animals↗
PubMed1987

Critical assessment of four monoclonal antibodies reactive with B-cells in formalin-fixed paraffin-embedded tissues.

Four commercially available monoclonal antibodies, MB1, MB2, LN1 and LN2, were studied to determine their sensitivity and specificity for the diagnosis of B-cell lymphomas when used on formalin-fixed paraffin-embedded tissues. In addition to 125 cases of immunologically characterized non-Hodgkin's lymphoma, a range of normal tissues, reactive lymphoid proliferations, Hodgkin's disease and granulocytic sarcomas were also studied. MB1 was found to give positive results in 53.6% of B-cell lymphomas, but the staining was sometimes weak and patchy; there was also cross-reaction with 1.8% of T-cell lymphomas. MB2 reacted with 88.4% of B-cell lymphomas and the reaction was often strong and diffuse, but it showed cross-reaction with 18.2% of T-cell lymphomas. LN1 and LN2 gave positive staining of 44.9 and 46.4% of B-cell lymphomas respectively, and the results appeared to be inferior to that obtained in B5-fixed tissues; staining was sometimes weak and focal, and they also gave false-positive results in a few cases of T-cell lymphoma. This study shows that MB1, LN1 and LN2 are fairly but not entirely specific for B-cells in the non-Hodgkin's lymphomas, but are not very sensitive when applied to formalin-fixed tissues. MB2 shows a high sensitivity but only moderate specificity. Therefore, when these antibodies are used to determine the immunophenotype of malignant lymphomas, the B-cell nature can be predicted with great confidence only when two, preferably three or more, of the antibodies give positive results. The potential applications of these antibodies are discussed.

Antibodies, Monoclonal↗
PubMed1993

Molecular diagnosis of trisomy 18 using DNA recovered from paraffin embedded tissues and possible implications for genetic counselling.

Severe malformations pointing to trisomy 18 were observed in two fetuses. No chromosome analysis had been performed. To confirm the diagnosis of trisomy 18, DNA was isolated from formalin fixed and paraffin embedded tissues. Molecular analysis by typing chromosome 18-specific (CA)n-repeats unequivocally showed the presence of trisomy 18 in both cases.

Chromosomes, Human, Pair 18↗
PubMed2000

A novel and single step ultrastructural method for staining biological tissues embedded in common embedding media.

A new single step staining method is described which is very simple, rapid and reproducible with better contrast than the widely employed uranyl acetate-lead citrate double staining method for observation of cell organelles in various tissue sections using transmission electron microscope. A copper uranyl acetate solution was employed to stain the ultrathin sections of tissues embedded in either water-miscible or water-immiscible resin media. The staining results are excellent. Cell organelles and extracellular matrix are stained with no artifactual precipitation or granulation.

Animals↗
PubMed2000

[Paraffin-embedded tissues as a source of control DNA].

During elucidation of crime cases, the need of a sample of DNA of the victim of the violent attack appears very often. Regarding to the obligatory archiving of histopathological preparations from sectioned persons, the paraffin-embedded tissues are easily achievable material suitable for the given purpose. Such a modification of an isolation method was elaborated which allows the yield of the sufficient amount of DNA from one histological section, and its usefulness was tested on tissues of different types and from different persons. The method was used successfully during solving of concrete cases.

DNA↗