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At least 1,441 records · Page 80Linked to original sources
PubMed2001

c-Kit (CD117) reactivity in extramedullary myeloid tumor/granulocytic sarcoma.

CONTEXT: c-kit, a proto-oncogene, encodes the transmembrane tyrosine kinase receptor CD117 and is detected by flow cytometry in the majority of cases of acute myeloid leukemia. The prognostic significance of the presence of c-Kit in acute myeloid leukemia is debated. Recently, c-kit inhibitors have been studied as possible therapies against hematopoietic malignancies; therefore, c-Kit detection may have important implications for treatment. OBJECTIVES: In this study, we investigated the expression of c-Kit in granulocytic sarcoma (GS) using paraffin-embedded tissue. DESIGN: Routinely formalin-fixed, paraffin-embedded tissues from 30 cases of GS were studied using immunohistochemistry. c-Kit (C-19) (a polyclonal antibody against carboxy terminal domain of c-Kit p145 or CD117) reactivity was compared with myeloperoxidase and lysozyme. The immunohistochemical panel also included CD34, CD68, CD43, Bcl-2, CD45RB, CD20, CD3, CD10, terminal deoxynucleotidyl transferase (TdT), and CD79a. RESULTS: The morphologic patterns included well-differentiated (5 cases), poorly differentiated (19 cases), and blastic forms (6 cases). Clinical data were obtained from 28 of 30 patients. Granulocytic sarcoma presented in lymph nodes in 10 cases, whereas in 20 cases it presented in extranodal sites. c-Kit reactivity was found in 87% (26/30) of the GS cases. There was no significant difference in c-Kit positivity between the nodal (90%, 9/10) and extranodal (85%, 17/20) neoplasms. c-Kit expression was not associated with the degree of the myeloid maturation. Two of 13 lymphoblastic lymphoma control cases and 1 of 28 of the large B-cell lymphomas were weakly immunoreactive with c-Kit. CONCLUSIONS: c-Kit reactivity can be demonstrated in a high percentage of GS cases; its presence may be useful not only in diagnosis, but also in the treatment of GS with new modalities.

Adolescent↗
PubMed1998

[Single-tube nested polymerase chain reaction technique in detecting Mycobacterium tuberculosis DNA].

OBJECTIVE: To study the sensitivity and specificity of single-tube nested polymerase chain reaction (SN-PCR) technique in detecting Mycobacterium tuberculosis DNA in paraffin-embedded tissues. METHOD: BCG DNA and the paraffin-embedded tissues of 30 cases with typical tuberculous lymphadenitis were examined to detect the IS6110 specific insertion sequences DNA of Mycobacterium tuberculosis complex by general PCR(G-PCR), double-tube nested PCR (DN-PCR) and SN-PCR techniques. RESULT: Fifteen fg or more BCG DNA could show positive results by DN-PCR and SN-PCR techniques, while 480 fg by G-PCR method. The positive rates of G-PCR, DN-PCR, and SN-PCR in detecting Mycobacterium tuberculosis DNA in 30 cases with tuberculous lymphadenitis were 43%, 100%, and 100% respectively, all of which were significantly different (P < 0.01) from that of the acid-fast staining method (10%). Significant differences in the positive rates also existed between G-PCR and DN-PCR, G-PCR and SN-PCR (both P < 0.01), while the positive rates of DN-PCR and SN-PCR were found same. CONCLUSION: The sensitivities of nested PCR techniques in detecting Mycobacterium tuberculosis are significantly higher than that of G-PCR, whereas the sensitivities as well as the specificities of SN-PCR and DN-PCR are the same. Thus SN-PCR seems more practicable.

DNA, Bacterial↗
PubMed2002

[Tissue microarrays, or the advent of chips in pathology].

Tissue microarray technology is a new method used to analyze several hundred tumor samples on a single slide allowing high throughput analysis of genes and proteins on a large cohort. The method consists to core tissues from paraffin-embedded tissue donor blocks and placing them into a single paraffin block. Despite the low amount of tissue analyzed by tissue microarray, different studies have demonstrated a high concordance of protein expression between this technique and the conventional tissue sections.

Biomarkers, Tumor↗
PubMed1990

Analysis of endocrine active and clinically silent corticotropic adenomas by in situ hybridization.

The distribution of pro-opiomelanocortin (POMC) messenger RNA (mRNA) in 7 functional and 17 clinically silent corticotropic adenomas was analyzed by in situ hybridization (ISH) with 35S-labeled oligonucleotide probes using formalin-fixed paraffin-embedded tissue sections cut from blocks that were in storage between 1 to 14 years. All 7 functional adenomas and 4 subtype 1 tumors had detectable POMC mRNA, while 3 of 6 subtype 2 and 1 of 7 subtype 3 silent adenomas contained detectable POMC mRNA. In situ hybridization analysis with an 35S-labeled beta-actin probe showed a positive hybridization signal in 22 of 22 cases, indicating that the absence of detectable POMC mRNA in some adenomas was not due to loss of the mRNAs during processing of the tissues or because of the age of the embedded tissue blocks. Northern hybridization analysis with the oligonucleotide probes in 2 normal pituitaries and an adenoma causing Cushing's disease detected a 1.2-Kb mRNA in all three tissues, indicating that the oligonucleotide probes were very specific. These results indicate that subtype 1 silent adenomas and clinically active adenomas associated with Cushing's disease contain POMC mRNA that is readily detectable by ISH in routinely processed tissue specimens, while only a few of the subtypes 2 and 3 adenomas have POMC mRNA that can be detected in paraffin blocks with the oligonucleotide probes used in this study.

Adenoma↗
PubMed1999

p53 immunoreactivity and single-strand conformational polymorphism analysis often fail to predict p53 mutational status.

The intent of this study was to investigate the ability of p53 expression and single-strand conformational polymorphism analysis (SSCP) to predict p53 mutational status in archival, paraffin-embedded tissues of gastric cancer. We evaluated paraffin-embedded tissues from 78 patients with advanced gastric cancer. The mutational status of the p53 gene (exons 5-9) was examined by SSCP analysis and by direct sequencing. These results were compared with p53 expression as assessed by immunohistochemical analysis (IHC). We graded p53 expression on a scale from 0 to 8 on the basis of both the intensity and the number of cells staining. Overall, we detected p53 immunoreactivity in 75.6% of the gastric cases; 19 (32.2%) of these cases scored from 1 to 4, and 40 (67.8%) cases scored from 5 to 8. p53 gene mutations were detected in 18 cases (23.1%) by SSCP and in 28 cases (36%) by direct sequencing. Thus, SSCP failed to detect 38% of the mutations found by sequencing. The majority of missed mutations involved exons 7 and 8. The concordance between IHC and SSCP was 37%, and the concordance between IHC and direct sequencing was 50%. Forty-five percent of cases positive by IHC failed to show mutations in exons 5 through 9. Five percent of cases negative by IHC (4 cases) contained mutations. One had a 1-base pair insertion; one had a mutation that resulted in a stop codon; the third had a mutation in exon 8; and the fourth had a mutation in both exons 5 and 8. Our findings indicate that p53 immunoreactivity correlates with the presence or absence of gene mutations in 50% of advanced gastric cancers when exons 5 through 9 are examined and that IHC cannot be reproducibly used as a marker of mutation in the most commonly mutated exons of the p53 gene. Furthermore, the sensitivity of SSCP for detecting mutations is only 62%. Thus, SSCP analysis cannot be used reliably to screen for p53 mutations.

Base Sequence↗
PubMed1992

A modified Da Fano silver stain for demonstration of neurons and dendrites in glycol methacrylate-embedded brain tissue.

Golgi impregnation techniques are commonly used for characterization of neurons and their dendritic and axonal processes. Most of the widely used techniques require processing of fresh brain tissues, which limits the amount of material available for study. Additionally, the stained blocks must be subsequently embedded in paraffin, which produces considerable cellular shrinkage and distortion artifacts. Modification by one investigator of an early silver impregnation technique, designed to demonstrate the Golgi apparatus, allowed demonstration of neurons and their dendritic processes. Our further modification of the later technique, along with embedding of the stained tissue in glycol methacrylate, permits detailed examination of neurons and their processes in formaldehyde-fixed neonatal human brains. In cerebellar sections, this modified technique impregnates nearly all Purkinje cells, elucidating the fine structural detail of the developing neuronal dendritic tree and spines.

Brain↗
PubMed1997

Quantitative analysis of mitochondrial DNA deletion in paraffin embedded muscle tissues from patients with KSS and CPEO.

The percentage of common deletion of mitochondrial DNA (mtDNA) was determined quantitatively by a PCR-based, non-radioactive method in DNA extracted from formalin-fixed, paraffin-embedded skeletal muscle tissues from two patients with Kearns Sayre syndrome (KSS) and one with chronic progressive external ophthalmoplegia (CPEO). The method involved PCR cycle titration of wild-type and deleted mtDNA in parallel, staining of gel bands with the sensitive fluorescence dye SYBR Green I, and quantitation of intensity on a computer screen by the NIH image program. We determined 75% and 71% common deletion of mtDNA in the KSS patients and 35% in the CPEO patient.

Adult↗
PubMed1996

Retrieval of leukocyte antigens in paraffin-embedded rat tissues.

We compared the immunohistochemical reactivity of various rat leukocyte antigens in frozen and paraffin-embedded thymus, spleen, abdominal lymph node, liver, and brain tissues of healthy Sprague-Dawley rats, fixed in various fixatives. Immune reactivity after fixation in Methacarn was superior to that of 4% neutral buffered formalin, a mercury-based fixative (B-5), or Carnoy. Microwave (MW) antigen retrieval (AR) enhanced antigen reactivity. Ten of the 11 leukocyte antigens studied could be retrieved in Methacarn-fixed, paraffin-embedded sections with a reactivity comparable to that obtained on frozen sections.

Animals↗
PubMed2006

Detection of West Nile virus in formalin-fixed, paraffin-embedded human tissues by RT-PCR: a useful adjunct to conventional tissue-based diagnostic methods.

BACKGROUND: West Nile virus (WNV), a member of genus Flavivirus, causes febrile illness, encephalitis, meningitis, myelitis, and occasional deaths in humans. Although several reverse transcription-polymerase chain reaction (RT-PCR) assays have been developed for detection of WNV in serum, cerebrospinal fluid, and fresh tissues, the usefulness of WNV RT-PCR assays for RNA extracted from formalin-fixed human tissues has not previously been demonstrated. OBJECTIVE: The objective of this study was to evaluate the application of a RT-PCR technique for the detection of WNV in routinely processed, formalin-fixed, paraffin-embedded (FFPE) human tissues, and to compare it with conventional serology and immunohistochemistry (IHC). STUDY DESIGN: We performed two WNV-specific nested RT-PCR assays targeting the viral capsid, premembrane, and envelope genes in FFPE central nervous system tissue samples from 27 patients with fatal WNV encephalitis, as confirmed by serology or IHC, and compared the results. The presence of WNV in RT-PCR-positive samples was confirmed by amplicon sequencing. RESULTS: Twenty (74%) patients were WNV RT-PCR positive while 24 (89%) were seropositive. WNV IHC staining of neurons and neuronal processes was positive in fourteen (52%) patients. The concordance between IHC and serology was 41% (11/27) and between RT-PCR and serology was 63% (17/27). All 11 seropositive/IHC-positive patients and 6 (46%) of 13 seropositive/IHC-negative patients were RT-PCR positive while all 3 seronegatives were positive by both IHC and RT-PCR. CONCLUSIONS: In this study, RT-PCR was significantly more sensitive than IHC in detecting WNV infections and provided specific sequence information about the infecting virus. RT-PCR on FFPE tissues may be a particularly useful diagnostic tool in patients who die relatively soon after disease onset and for whom serology may be negative. Combined use of serology, IHC, and RT-PCR would be expected to have the best overall sensitivity and improve detection of fatal WNV infection.

Adult↗
PubMed2005

[Desmoplastic small round cell tumor: a clinicopathologic study of 15 cases].

OBJECTIVE: To study the clinicopathologic features and immunophenotype of desmoplastic small round cell tumor (DSRCT), and to assess the feasibility of reverse transcriptase-polymerase chain reaction (RT-PCR) as a diagnostic adjunct for DSRCT in routine practice. METHODS: The clinical (number = 15), cytologic (number = 1) and histopathologic (number = 14) features of 15 cases of DSRCT were investigated. The immunophenotype was studied by LSAB method using a panel of antibodies. RT-PCR was performed in one case using formalin-fixed, paraffin-embedded tissue for EWS-WT1 fusion gene mRNA. RESULTS: Among the 15 patients studied, 13 were males and 2 were females. Their age ranged from 12 to 38 years (mean age = 23.8 years). Most presented with vague abdominal discomfort, distension or pain, accompanied by nausea, constipation and weight loss. Physical examination showed a palpable abdominal mass with ill-defined borders and tenderness. Ultrasound and computerized tomographic examination revealed single or multiple nodular tumor mass(es) in the peritoneal cavity, measuring 3 cm to 25 cm in greatest diameter (mean tumor diameter = 8.6 cm). Cytologic examination in 1 case showed clusters of small round cells in a hemorrhagic background. The tumor nuclei were hyperchromatic and contained inconspicuous nucleoli. Mitotic figures were readily identified. The cytoplasm however was scant. Histologically, the tumor was composed of small, round, ovoid to spindled cells arranged in nests of various shapes and sizes, embedded in a desmoplastic and focally hyalinized stroma. Immuno- histochemically, all cases showed diffuse and strong staining for AE1/AE3, vimentin, desmin and neuron-specific enolase. Some of them also expressed CAM5.2, epithelial membrane antigen, CD57, chromogranin A, synaptophysin and WT1. They were all negative for myogenin, CK5/6, CD117, calretinin and CD99. RT-PCR successfully amplified the EWS-WT1 chimeric mRNA in 1 case using paraffin-embedded tissue. Subsequent DNA sequencing showed that the gene fusion involved exon 7 of EWS and exon 8 of WT1 genes. The fusion gene contained KTS sequence. CONCLUSIONS: DSRCT is a highly malignant small round cell tumor occurring predominantly in the abdominal or pelvic cavity of young to middle-aged males. It is characterized by multiphenotypic differentiation. The peculiar perinuclear dot-like staining pattern for vimentin and desmin is characteristic for DSRCT. EWS-WT1 fusion transcript can be detected in formalin-fixed, paraffin-embedded tissue by RT-PCR, which may thus serve as a useful diagnostic adjunct for DSRCT.

Abdominal Neoplasms↗
PubMed1995

[Detection of enteroviral RNA in paraffin-embedded myocardial tissue from patients with Keshan by nested PCR].

Enteroviral RNA in paraffin-embedded myocardial specimens from 21 patients with Keshan disease and 21 patients with cerebral trauma was investigated by nested polymerase chain reaction. Of the 21 patients with Keshan Disease, 18 (85.7%) showed positive findings. Their myocardial tissues were infected by enterovirus previously. The results suggest that enterovirus infection is the major cause of Keshan disease in the Chuxiong district, Yunnan province.

Base Sequence↗
PubMed1988

Immunogold localization of peroxisomal enzymes in Epon-embedded liver tissue. Enhancement of sensitivity by etching with ethanolic sodium hydroxide.

Epon-embedded biological materials exhibit well preserved ultrastructure but generally weak antigenicity. Brief etching of ultrathin Epon sections with resin solvent, ethanolic sodium hydroxide, brought about a nearly 2-fold increase in the immunogold labeling density of rat and human hepatic peroxisomes after incubation with antisera against catalase and 3 enzymes of lipid beta-oxidation: acyl-CoA oxidase, bifunctional protein (enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase) and 3-ketoacyl-CoA thiolase. The etching was superior to pretreatment with an oxidant, sodium metaperiodate. Despite some deterioration of the cellular ultrastructure, the obtained enhancement of the sensitivity of the protein A-gold method may be helpful in cases when the antigenicity to be detected is strongly inhibited by epoxy resin embedding.

Acetyl-CoA C-Acyltransferase↗
PubMed1994

Effect of formalin fixation and long-term storage on the detectability of bovine viral-diarrhoea-virus (BVDV) RNA in archival brain tissue using polymerase chain reaction.

Detection of DNA or RNA in formalin-fixed, paraffin-embedded tissues using polymerase chain reaction (PCR) may be hindered by degradation of nucleic acids during tissue collection, preparation and archivation. This study describes investigations on the effect of formalin fixation and prolonged storage of paraffin-embedded tissues on bovine viral-diarrhoea (BVD)-virus RNA as a model system. Brain tissues from eight persistently BVDV-infected calves containing high amounts of the virus were fixed in 5% neutral-buffered formalin or 10% non-buffered formalin for different fixation times, respectively, and paraffin embedded. Subsequent detection of an 803 bp fragment from single tissue sections using nested PCR after reverse transcription (nested RT-PCR) demonstrated a loss of detectability of viral RNA after more than 10 days (10% non-buffered formalin) and 3 months (5% neutral-buffered formalin) of fixation. Additional studies with 280 initially BVDV-positive brain tissues from 25 persistently BVDV-infected calves after storage of up to 10 years revealed a loss of detectable RNA after more than 1 year of storage. For estimation of the higher sensitivity of nested RT-PCR compared to single step RT-PCR, serially diluted BVD virus suspensions were examined using both methods. Nested RT-PCR was found to be about 100-fold more sensitive than single-step RT-PCR, and is therefore recommended as the appropriate technique for archival studies.

Animals↗
PubMed1985

Germ cell quantitation in human testicular biopsy.

Quantitative analysis of human seminiferous epithelium was carried out using an improved method of glutaraldehyde and osmium fixation with plastic embedding. Part of each biopsy specimen was fixed in Bouin's fixative and embedded in paraffin for comparison. Epon embedded tissue had very little artifactual damage compared with paraffin embedded tissue sections. The germ cell to Sertoli cell ratios were determined by counting the various germ cells per "unit" tubular area. Data obtained by this method reflect a remarkable stability of Sertoli cell number and germ cell-Sertoli cell ratios both between biopsies from different individuals and between biopsies from right and left testes from the same individual. Agreement between the present results and those of earlier studies based on paraffin embedded testicular specimens supports the validity of this method of germ cell quantitation of human testicular biopsy samples.

Adolescent↗
PubMed1990

Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.

Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase K was required to produce good yields of DNA. Incubation with sodium dodecyl sulphate produced very poor yields, while boiling produced 20% as much DNA as long enzyme digestion. DNA extracted by these methods was suitable for the PCR amplification of a single copy gene. Proteinase K digestion also produced considerable amounts of RNA which has previously been shown to be suitable for PCR analysis. A delay before fixation had no effect on the amount of DNA obtained while fixation in Carnoy's reagent results in a much better preservation of DNA than formalin fixation, allowing greater yields to be extracted.

DNA↗
PubMed2006

Uterine carcinosarcoma: immunohistochemical studies on tissue microarrays with focus on potential therapeutic targets.

OBJECTIVE: Carcinosarcoma of the uterus is a highly aggressive tumor containing both malignant epithelial and spindle (mesenchymal) components. Because of their rarity and poor clinical outcome, investigations into the expression of potential therapeutic targets are limited. The aim of this study was to determine the expression of therapeutic targets in both the epithelial and spindle (mesenchymal) components in 30 carcinosarcomas using tissue microarrays, for potential treatment strategy. METHODS: We collected formalin-fixed, paraffin-embedded tissue blocks of carcinosarcoma of the uterine corpus resected from 30 patients who had undergone total abdominal hysterectomies at our institution between 1985 and 2005 (ages 38-83 years, mean 65.9 years). All hematoxylin-eosin stained sections from each tumor were reviewed to confirm the pathologic diagnosis. Two tissue cores from the paraffin-embedded tissue blocks were constructed into a tissue microarray. Sections were stained with monoclonal antibodies against HER-2, VEGF, c-KIT, COX-2, and EGFR. Unequivocal staining of at least 5% tumor cells was considered positive. HER-2 amplification was also examined by fluorescence in situ hybridization (FISH) in 2 cases. RESULTS: In the epithelial component, expression of HER-2, VEGF, c-KIT, COX-2, and EGFR were detected in 2 (6%), 30 (100%), 0 (0%), 21 (70%), and 9 (30%) cases, respectively, whereas these expressions in the spindle (mesenchymal) component were detected in 0 (0%), 28 (93%), 0 (0%), 5 (16%), and 20 (67%) cases, respectively. By FISH, one of the two cases with HER-2 expression showed gene amplification (2.62). CONCLUSIONS: VEGF is strongly expressed in both the epithelial and spindle (mesenchymal) components of uterine carcinosarcoma. This result warrants further study to evaluate the possible role of anti-angiogenic agents in cancer therapy for patients with uterine carcinosarcomas. The expression patterns of COX-2 and EGFR differed between the epithelial and spindle (mesenchymal) components. HER-2 and c-KIT are poor therapeutic targets for uterine carcinosarcomas.

Adult↗
PubMed2000

Quantitation of DNA extracted after micropreparation of cells from frozen and formalin-fixed tissue sections.

Quantitation of DNA from microdissected fresh-frozen or paraffin-embedded tissue sections would be not only a valuable tool for ensuring optimum reaction conditions for many types of qualitative polymerase chain reaction (PCR) analyses, but also a prerequisite for any kind of subsequently performed genetic analyses aimed at the absolute quantitation of target sequences. The present study describes the quantitation of DNA after microdissection and extraction of cells with the PicoGreen fluorescence method. The limits of detection and of quantitative determination, respectively, have been determined by measuring dilutional series of three different DNA extractions, using either a medium-scale preparation from a solid tissue specimen or a known number of leukocytes or microdissected cells from frozen tumor sections. As corresponding limits of detection, 26, 24, and about 40 diploid genomes, and as limits of quantitative determination, 80, 73, and about 120 diploid genomes were obtained. Furthermore, it was shown that formalin fixation as well as hematoxylin staining of frozen sections with Delafield's and Mayer's alum or Weigert's iron hematoxylin before microdissection significantly diminishes the amount of extractable DNA and may lead to less reliable results, even of qualitative PCR analysis. In conclusion, the PicoGreen method allows precise quantitation of DNA corresponding to a minimum of about 120 diploid cells. It provides the basis for reliable qualitative analyses as well as the precondition for further quantitative genetic measurements from microdissected frozen or formalin-fixed and paraffin-embedded tissue sections.

DNA, Neoplasm↗
PubMed1988

Antigen localization in immunoperoxidase-stained plastic-embedded soft tissues.

Immunoperoxidase stains were performed on normal and neoplastic tissue from prostate, colon, thyroid, lung, nerve, uterus, and placenta embedded in both plastic (glycolmethacrylate [GMA]) and paraffin. Positive results in plastic section were obtained for carcinoembryonic antigen (CEA), keratin, epithelial membrane antigen (EMA), thyroglobulins, S-100, prostate-specific antigen, human chorionic gonadotrophin (HCG), and beta-HCG. More delicate staining with more precise localization of antigens is noted. Superior (paraformaldehyde) fixation and cold processing followed by GMA polymerization (4 degrees C) allow for optimum antigen survival. After fixation, tissue processing involves a series of 0.1 mol/L phosphate buffer rinses with sucrose and ammonium chloride in a conventional dip-and-dunk processor placed in a 4 degrees C cold room. Acetone dehydrations are used before GMA infiltration, cold polymerization, and sectioning. Before immunoperoxidase staining, the plastic section is digested in .25% bovine trypsin for ten minutes. The immunoperoxidase methods described can be useful when small biopsies are routinely embedded in plastic to obtain improved histologic (hematoxylin-eosin) sections. There may also be research applications in quantifying antigen expression in benign, dysplastic, and neoplastic tissues by examining the stains under high power.

Adenocarcinoma↗