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At least 397 records · Page 22Linked to original sources
PubMed1994

Androgen receptor immunohistochemistry on paraffin-embedded tissue.

Androgen deprivation remains the primary therapy for patients with metastatic prostate cancer. Response to hormonal manipulation, however, has been quite variable. Androgen receptor (AR) expression has been used to predict clinical response to antiandrogenic treatment. However, methods of detection of AR expression have been limited to receptor biochemical assays in cytosolic or nuclear fractions of frozen tissue homogenates with obvious contamination problems by nonmalignant epithelial and stromal cells. As a result, studies correlating AR expression with response to hormonal therapy have been limited and controversial. More recently, immunohistochemical methods of analysis have become available, but only on frozen tissue. We describe a simple method to evaluate ARs on formalin-fixed, paraffin-embedded tissue sections using antigen retrieval methods. Primary, as well as metastatic, prostate carcinomas showed nuclear staining for ARs. Secretory cells stained uniformly in hyperplastic and normal prostatic glands. The majority of stromal cells had strong nuclear positivity. All other cancers tested (colon, breast, lung, skin, kidney) were negative. This immunohistochemical technique allows evaluation of prostate cancer AR status in routinely processed tissues. Thus, application of this technology to archival material should permit assessment of whether AR expression is predictive of response to endocrine therapy in advanced prostate cancer.

Adenocarcinoma↗
PubMed1997

In situ DNA fragmentation assay for detection of apoptosis in paraffin-embedded tissue sections. Technical considerations.

Detection, by light microscopy, of cells in situ undergoing apoptosis has been improved by use of an in situ apoptosis (DNA fragmentation) assay on formalin-fixed and paraffin-embedded tissue sections. We studied conditions of tissue preparation and fixation that may affect the test results. In this study, we intended to determine whether archival tissues prepared under unknown conditions can be used for the in situ apoptosis assay. All tissue sections were pretreated with Proteinase K, followed by incubation with biotinylated 11-deoxyuridine triphosphate in terminal deoxynucleotidyl transferase and then avidin-biotin-peroxidase complex. The following formalin-fixed and paraffin-embedded histologic sections were tested: (1) normal tissues from surgically resected specimens fixed immediately or stored at 4 degrees C and then fixed after 1, 2, 4, 6, or 24 hours; (2) archival autopsy material from histologically normal tissues; and (3) freshly prepared normal tissues from C57 mice. We observed that fixation- and prefixation-elapsed times do not adversely affect the results of the assay. Similar, if not identical results were seen in archival human tissues stored for up to 25 years, the normal tissues freshly prepared from surgical specimens, and the tissues from C57 mice. We conclude that the in situ assay of DNA fragmentation is rapid, sensitive, and reproducible. The use of formalin-fixed and paraffin-embedded archival material as old as 25 years opens the way for a variety of studies of apoptosis in diverse pathologic states.

Animals↗
PubMed1993

Loss of heterozygosity detected in formalin-fixed, paraffin-embedded tissue of colorectal carcinoma using a microsatellite located within the deleted in colorectal carcinoma gene.

We determined loss of heterozygosity from formalin-fixed, paraffin-embedded tissue of colorectal carcinoma using microsatellite polymorphism. The polymorphism was assayed based on DNA amplification by the polymerase chain reaction (PCR). The PCR-analyzed microsatellite method was applied to assay degraded DNA extracted from paraffin-embedded blocks with adenocarcinoma of colon. The DNA from 26 tumors as well as their corresponding normal tissue samples were successfully amplified using a dinucleotide microsatellite located within an intron of the deleted in colorectal carcinoma gene. Allele losses on this marker were detected in 33% of informative colorectal carcinomas. This study demonstrates that microsatellites provide a powerful set of DNA markers for loss of heterozygosity on archival specimens.

Adenocarcinoma↗
PubMed1992

An experience of cell sorting using samples isolated from paraffin-embedded tissue of neuroblastoma.

Flow cytometric DNA content analyses were performed on samples from 72 patients with neuroblastoma. Nuclear suspensions obtained from paraffin-embedded samples were prepared using the method described by Schutte et al. Determination of DNA ploidy was possible in 62 out of 72 patients. DNA diploidy was detected in 23 (37.1%) and DNA aneuploidy was found in 39 (62.9%) of these 62 patients. Cell sorting was performed on two peak areas of the DNA histogram of samples with aneuploidy. The sorted cells of the first peak area were composed of small round cells and were considered to be normal lymphocytes. The sorted cells of the second peak area were composed of relatively large cells with obvious nucleoli and were considered to be neuroblastoma cells. Thus, two cell components could be distinguished by cell sorting. It is concluded that cell sorting may be of benefit in giving detailed information about DNA histograms, even in samples obtained from paraffin-embedded tissue.

Cell Separation↗
PubMed1994

Immunohistochemical evaluation of myeloid leukemia infiltrates (granulocytic sarcomas) in formaldehyde-fixed, paraffin-embedded tissue.

Paraffin sections of granulocytic sarcomas (GS) (n = 30) were immunohistochemically evaluated for CD3, CD15 (LeuM1), CD20 (L26), CD31, CD34, CD43, CD45, CD68 (KP1), lysozyme, myeloperoxidase (BM1), CD45RO (UCHL1), and LN5 with an avidin-biotin amplification system and a peroxidase-based color development system with DAB as a chromogen. CD45 positivity was present in all lymphomas and 24 of 25 granulocytic sarcomas. Lysozyme and CD43 labeled 26 of 29 granulocytic sarcomas, showing intense cytoplasmic staining. LN5 (membrane-staining) and CD68 (subtle cytoplasmic caplike staining) were found in 20 of 30 cases, often only focally. BM1 and CD15 mainly labeled maturing granulocytes and mostly were negative in primitive myeloid cells. Myeloid progenitor cell antigens CD31 and CD34 were seen in 7 and 12 of 30 cases, respectively. They seemed to recognize different subsets of myeloid leukemia infiltrates (16 cases positive for at least one); the use of CD31 and CD34 for defining these subsets should be evaluated further. Features suggesting a dual phenotype--T-cell and myeloid (positive for CD3, CD68, and lysozyme)--were documented in two cases. In contrast, lymphoblastic lymphomas (n = 4) were positive for CD3 and CD43 but negative for CD68, lysozyme, CD31, CD34, LN5, and myeloperoxidase. Lymphocytic lymphomas (n = 10) were positive for CD20 and CD43 but negative for all other markers. Small, round-cell tumors (n = 15) were negative for all markers. If T-cell and B-cell differentiation can be excluded with other markers, CD43+ is a sensitive marker for myeloid differentiation. Our results show that several markers are useful in the identification of myeloid leukemia infiltrates and in distinguishing them from lymphoblastic and lymphocytic lymphomas and small round-cell tumors in formaldehyde-fixed, paraffin-embedded tissue.

Adult↗
PubMed1991

PCR amplification from paraffin-embedded tissues: recommendations on fixatives for long-term storage and prospective studies.

The development of polymerase chain reaction (PCR) DNA amplification methods has afforded molecular studies of fixed paraffin-embedded tissue samples and other archival material. Some fixation methods damage DNA, and thus deleteriously affect subsequent PCR analysis. This study addressed the effect of short- and long-term storage (2 hr to 30 days) in a variety of fixatives (10% buffered-neutral formalin [BNF], 95% ethanol, acetone, and OmniFix) before paraffin embedding. We tested the ability of prepared tissue sections to yield DNA amplification products ranging from 268 to 1327 bp. Results indicated that tissues fixed for 8 days in BNF were able to amplify 536-bp but very little 989-bp DNA fragments; after 30 days of BNF fixation only a 268-bp fragment was amplifiable. Samples fixed in OmniFix and acetone yielded products of 989 and 1327 bp, respectively, after 8 days of fixation; both fixatives yielded 989-bp amplification products after 30 days of fixation. Tissues fixed in 95% ethanol for up to 30 days efficiently produced DNA amplification fragments of up to 1327 bp in length. The results provide important information for prospective studies that involve PCR analysis from archival material. Furthermore, fixation and long-term storage in ethanol should prove particularly useful in remote areas where refrigeration or immediate sample-processing is unavailable.

DNA↗
PubMed1989

Primary invasive and in situ vaginal carcinoma. Flow cytometric analysis of DNA aneuploidy and cell proliferation from archival paraffin-embedded tissue.

Eleven invasive and five non-invasive primary vaginal carcinomas were studied by DNA flow cytometry using archival paraffin-embedded tissue as starting material. Overall frequency of DNA aneuploidy in the invasive carcinomas was 8/11 (73%). DNA aneuploidy occurred in all four advanced stage (III-IV) and in 4/7 (57%) of the early stage (I-II) carcinomas. Among the squamous cell carcinomas aneuploid DNA content was also associated with non-keratinizing tumor type. Invasive vaginal carcinomas showed a high median S-phase fraction (SPF) (18.4%, range 6.9-31.8%). High SPF values were associated with advanced stage and non-keratinizing tumors. Corrected 5-year survival rate in invasive vaginal cancer was 44%, with no significant relation to DNA ploidy or SPF. In situ carcinomas were almost as often DNA-aneuploid (3/5, 60%) as the invasive carcinomas and had comparable median SPF value (13.4%, range 5.5-24.6). One in situ carcinoma with a high DNA-index and SPF relapsed, but overall 5-year survival rate was 100%. In conclusion, both invasive and in situ vaginal carcinomas frequently contain DNA-aneuploid stemlines and show a high SPF. Although DNA aneuploidy and high SPF correlate with advanced stage and non-keratinizing tumor type, they do not have much prognostic relevance in vaginal neoplasia.

Aneuploidy↗
PubMed1988

Nuclear DNA content in prostatic carcinoma measured by flow cytometry: a retrospective study on paraffin-embedded tissue.

The nuclear DNA content in 67 prostatic carcinomas was measured by flow cytometry of nuclear suspensions prepared from paraffin-embedded tissue blocks. A method was developed that provided nuclear suspensions of good quality whether tissue blocks from transurethral resections or transvesical prostatectomies were used. Thirty (43.7%) of the tumors were found to be aneuploid. No significant correlation between clinical stage or histological grade and aneuploidy was demonstrated.

Aged↗
PubMed1985

Extraction of cells from paraffin-embedded tissue sections for single-cell DNA cytophotometry.

A simple technique is presented for the isolation of cells from paraffin-embedded tissues for Feulgen DNA cytophotometric investigations. Tissue fragments from paraffin blocks were deparaffinized in xylene, rehydrated and refixed in a formalin solution and incubated in a solution of 0.5 pepsin in 0.25% hydrochloric acid. After filtration through a 70 micron mesh and centrifugation, the cells were smeared upon a glass slide. Comparison between the results obtained with freshly prepared imprints and with pepsin-extracted cells of the same tumor showed that the extraction technique does not influence the Feulgen reaction or the DNA distribution pattern. Investigations carried out on bladder and embryonal carcinomas have demonstrated that the method permits an analysis of histologically or histochemically identified tumor cells within individual tissue areas.

Cell Separation↗
PubMed1994

Enhancement of hybridization efficiency in interphase cytogenetics on paraffin-embedded tissue sections by microwave treatment.

To enhance the efficiency of in situ hybridization in interphase cytogenetics on formalin-fixed and paraffin-embedded tissue sections we studied the effect of microwave heating as an addition to tissue pretreatment. The modified protocol resulted in a reduction of proteolytic digestion time and of effective enzyme concentration. Apart from that, reproducibility was raised and in large sections successful hybridization was more homogenous. We conclude, that the given technical recommendations should be useful for the study of chromosomal aberrations in routinely processed tissues.

Chromosome Aberrations↗
PubMed1986

Pararosaniline or acriflavine-Schiff staining of epoxy embedded tissue after periodic acid oxidation in ethanol: a method suitable for morphometric and fluorometric analysis of glycogen.

A method for preparing tissue sections for automatic image analysis of glycogen is described. Large semithin sections of epoxy embedded tissue fixed in glutaraldehyde-osmium were stained with Schiff reagent and acriflavine (fluorescent staining) after resin removal and periodic acid oxidation in ethanol. We found it essential to avoid tissue rehydration before final staining. The Schiff stain permits an assessment of the cellular volume of glycogen, and the acriflavine allows a fluorometric evaluation of glycogen density.

Acriflavine↗
PubMed1995

Interphase cytogenetic analysis of single cell suspensions prepared from previously formalin-fixed and paraffin-embedded tissues.

Fluorescence in situ hybridization (FISH) provides a rapid and accurate method for the detection of chromosomal aneuploidy. We have developed a technique for the use of FISH on single cell suspensions produced from either formalin-fixed or paraffin-embedded tissues. Preparation of such tissues involves sequential rehydration, enzymatic digestion to release single nuclei, and hybridization with a fluorescently labeled chromosome-specific centromeric probe. In a clinical setting formalin-fixed tissue from many tissue types is readily available for additional retrospective study. FISH on formalin-fixed tissues is especially beneficial in follow-up studies of cases involving termination after prenatal diagnosis or patients with a malignant disease where previous routine cytogenetics established the chromosomal aneuploidy. The use of this technique eliminates the biases of cytogenetic analysis due to clonal selection in tissue culture, the low number of cells analyzed, and the restriction to only dividing cell populations. We have demonstrated that this application of interphase cytogenetics to the study of various formalin-fixed tissues is amenable to the detection of chromosomal aneuploidies and has specific advantages over cytogenetic analysis.

Aneuploidy↗
PubMed1995

Detection of clonal lambda light chain gene rearrangements in frozen and paraffin-embedded tissues by polymerase chain reaction.

A method was established to detect clonal lambda light chain gene rearrangements in peripheral blood lymphocytes and frozen or paraffin-embedded tissues. V lambda-gene-family-specific primers were used together with a J lambda primer mix in separate reactions to amplify V lambda gene rearrangements by the polymerase chain reaction. Clonal lambda gene rearrangements were detected in seven of seven lambda-expressing B cell leukemias, in four of five lambda-expressing non-Hodgkin's lymphomas with frozen tissues, and in seven of nine cases of lambda-expressing non-Hodgkin's lymphomas for which formalin-fixed, paraffin-embedded specimens were available. Clonality of amplified polymerase chain reaction products was confirmed by sequence analysis for several cases. The present study shows that it is possible to amplify clonal lambda gene rearrangements in the majority of lambda-expressing B cell leukemias and lymphomas. The method described here, therefore, is a useful supplement to the previously described approach of VH and VK gene amplification to detect clonal B cell populations and allows the study of V lambda gene usage and somatic mutation in lambda-expressing normal and malignant B cells.

Amino Acid Sequence↗
PubMed1994

A simple, rapid method for isolating RNA from paraffin-embedded tissues for reverse transcription-polymerase chain reaction (RT-PCR).

Described here is a simple, rapid technique for isolating RNA from archived formalin-fixed, paraffin-embedded tissues (PETs). Using this modified acid guanidinium thiocyanate method, RNA sufficient as a template for the reverse transcription-polymerase chain reaction (RT-PCR) can be isolated in 2 hr from a single 20-microns-thick section of tissue. Spliced mRNA corresponding to portions of the estrogen receptor (ER) gene was successfully amplified from fixed, embedded human breast cancers containing increased amounts of ER protein. This method makes it feasible to safely and efficiently isolate RNA from large numbers of routinely processed paraffin blocks for retrospective studies of endogenous proteins such as hormone receptors, oncogenes, and viruses.

Base Sequence↗
PubMedDate not supplied

A rapid and simplified technique for analysis of archival formalin-fixed, paraffin-embedded tissue by fluorescence in situ hybridization (FISH).

We here present a simplification of the entire procedure for preparing formalin-fixed, paraffin-embedded tissue to be used for FISH-analysis. The steps for deparaffinisation and disintegration of the tissue to produce intact cell nuclei in a monodispersed suspension are detailed as well as the hybridisation steps. The procedure results in a clear cell suspension with intact cell nuclei and without clumped debris particles. The enzymatic digestion is restricted to the trypsinisation step of the deparaffinisation procedure. No further pretreatments prior to hybridization are performed. This modified technique has been successfully applied to different formalin-fixed, paraffin-embedded materials.

Cell Nucleus↗
PubMed1996

Technical approach for the study of the genetic evolution of breast cancer from paraffin-embedded tissue sections.

We have optimized a technique that allows the study of numerous chromosomal loci (n = 20-50) from single paraffin-embedded tissue sections by microsatellite length polymorphism analysis. DNA samples from normal and breast cancerous tissue can be obtained from the same section by means of microdissection. This technique was further improved by subjecting DNA to several cycles of amplification with a degenerate (universal) primer and then with specific microsatellite primers. This amplified DNA was also used to screen for mutations in the p53 gene by means of PCR-SSCP. In addition adjacent tissue sections were used to assess specific chromosome copy number by interphase cytogenetic analyses (chromosome in situ hybridization) and to analyze expression of specific genes such as p53 and ERBB2. As an example of the use of our approach we performed a detailed chromosome 17 allelotypic analysis in 22 breast tumors (5 ductal carcinomas in situ, 13 invasive ductal carcinomas, and 4 invasive lobular carcinomas). We detected mutations in the p53 gene by PCR-SSCP in 36% of the samples. Samples with significant levels of p53 protein accumulation detected by immunohistochemistry were also positive for mobility shifts in the SSCP analysis. We observed that chromosome 17 allelic losses and imbalance occurred at as early a stage as ductal carcinoma in situ (DCIS). Although in some cases we observed allelic losses or imbalance affecting the 17p13 region, close to the p53 locus, several of the tumors showed dissociation between such loss or imbalance and p53 mutation. Lobular carcinomas were predominantly disomic for chromosome 17 in contrast with ductal tumors, which often showed polysomy for chromosome 17. This comprehensive approach correlating the tumor subtype, its allelotype, with specific chromosome copy number and specific gene mutations and expression in preinvasive or early invasive breast cancer lesions will potentially provide information of relevance for a better understanding of the multistep mechanisms of breast carcinogenesis.

Alleles↗
PubMed1997

DNA extraction from formalin-fixed, paraffin-embedded tissues: protein digestion as a limiting step for retrieval of high-quality DNA.

Several DNA extraction methods have been used for formalin-fixed, paraffin-embedded tissues, with variable results being reported regarding the suitability of DNA obtained from such sources to serve as template in polymerase chain reaction (PCR)-based genetic analyses. We present a method routinely used for archival material in our laboratory that reliably yields DNA of sufficient quality for PCR studies. This method is based on extended proteinase K digestion (250 micrograms/ml in an EDTA-free calcium-containing buffer supplemented with mussel glycogen) followed by phenol-chloroform extraction. Agarose gel electrophoresis of both digestion buffer aliquots and PCR amplification of the beta-globin gene tested the suitability of the retrieved DNA for PCR amplification.

Adrenal Glands↗
PubMed1978

A comparison of iron histochemical methods for use on glycol methacrylate embedded tissues.

Four histochemical tests for iron and four procedures for its removal were investigated in regard to their suitability for glycol methacrylate embedded tissues. The HCl-ferrocyanide and chlorate hematoxylin methods were easily modified for plastic sections. The latter does not use iron-containing reagents. Desiderization was complete both after a fifteen minute exposure in 1% Na2S2O4 in 0.1 M acetate-HCl buffer (pH 4.5) and, if an acid method is preferred, after twelve hours in 5% oxalic acid. A six hour treatment in 3.7 N H2SO4 also removed all histochemical iron but was accompanied by a relatively greater loss of tissue basophilia.

Animals↗