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At least 1,063 records · Page 59Linked to original sources
PubMed2004

Fluorescence in situ hybridization on formalin-fixed and paraffin-embedded tissue: optimizing the method.

Fluorescence in situ hybridization (FISH) is widely used to study numerical and structural genetic abnormalities in both metaphase and interphase cells. The technique is based on the hybridization of labeled probes to complementary sequences in the DNA or RNA of the cells. Interphase FISH is most often applied on cytologic material such as hematologic smears or imprints, but the method is also used to study genetic changes in tissue sections when morphology is important or when cytologic material is not available. In cases in which the presence of intact nuclei is of importance, such as quantitation of signals as in triploidy, it is possible to isolate nuclei from paraffin-embedded tissue. However, using formalin-fixed paraffin-embedded tissue, either in thin sections or as isolated nuclei, one encounters a range of technical problems, paralleling those met in immunohistochemistry. Variations in time lapse between removal of tissue and fixation, duration of fixation, enzymatic pretreatment, hybridization conditions, and posthybridization washing conditions are important factors in the hybridization. In this study, we have listed the results of a systematic approach to improve FISH on isolated nuclei and tissue sections from formalin-fixed, paraffin-embedded tissue.

Chromosome Aberrations↗
PubMed2006

The HOPE technique opens up a multitude of new possibilities in pathology.

Fixation of tissues with formalin results in well-preserved morphology but to a high degree leads to degradation of nucleic acids, which substantially constricts the spectrum of applicable molecular techniques. The novel HOPE-fixative with subsequent paraffin embedding, as an alternative to formalin, has been shown to result in a morphological preservation comparable to formalin-fixed, paraffin-embedded specimens. Due to a similar workflow like in formalin-fixation and paraffin embedding, the HOPE technique can be successfully established within any pathological institute. We have shown that DNA, RNA and proteins are protected in HOPE-fixed, paraffin-embedded tissues for at least eight years. Moreover, we described procedures which permit successful application of all common molecular techniques such as in situ hybridization targeting either DNA or RNA, immunohistochemistry without antigen retrieval and for formalin-refractory antigens, PCR, RT-PCR, Western blot, Northern blot, and transcription microarrays to HOPE-fixed, paraffin-embedded tissues. Furthermore, HOPE-fixed tissues can be used for the construction of tissue microarrays for enhanced high-throughput analyses on the molecular level. Using the HOPE technique as its crucial methodological base, ex vivo model systems could be established, e.g. for the simulation of early events in human infections and detection of chemotherapy resistances in human cancer. In addition to tissues, cell-culture preparations have been prepared utilizing the HOPE technique, which were then successfully applied to in situ hybridization targeting mRNA or immunocytochemistry with excellent preservation of morphological details. Taken together, the HOPE technique to date represents an alternative fixation that is, in contrary to other procedures, scientifically broadly analyzed. Therefore new possibilities are opened up especially within the rapidly growing field of molecular pathology.

Fixatives↗
PubMed2006

An investigation of the problem of two-layered immunohistochemical staining in paraformaldehyde fixed sections.

In sections of paraformaldehyde fixed brain tissue, stained using immunohistochemical methods, the distribution of staining within the sections is not uniform. Whilst stained cells are seen at the top and bottom surfaces, the central thicknesses of the sections contain little or no immunoreactivity. This presents a major problem for quantification, as each section contains a population of cells that is not visualized by the staining method. Following extensive investigation of this phenomenon, we report that the failure of full thickness, immunohistochemical staining is not a failure of the immunohistochemical methodology per se, nor is it related directly to the thickness of the sections used. Rather, the problem lies in the chemistry of the tissue itself, and originates during fixation of the tissues using paraformaldehyde-based perfusion methods, which render the cell membranes impermeable to one or more components of the staining protocol. We show that this impermeability is affected by addition of membrane-disrupting agents to the fixative, and by a reduction of exposure to paraformaldehyde during fixation. The present investigation contributes to the development of new fixation protocols, optimised for use in both immunohistochemical methods and morphometric analyses.

Adenoviridae↗
PubMed2005

Histologic assessment of mesh fixation following laser-assisted tissue soldering in a lapine model.

OBJECTIVE: Wound histology and mesh bioincorporation following intraperitoneal fixation using laser-assisted soldering was evaluated. METHODS: 2.8-3.2 kg NZW rabbits underwent laparotomy. Controls had 2x2 cm segments of Mersilene stapled to peritoneum. Group 2 segments were affixed with 55% collagen solder onlay by fiber-coupled diode laser (1.43 +/- 10 micro, 2.5 W CW, 4 mm spot, 60 degrees C set temperature). Group 4 had Mersilene inlaid into melted solder. Group 3 had solder-embedded Vicryl mesh affixed. Animals were euthanized at 0, 2, 4, 6 weeks. Fixed sections were assessed for integrity, inflammation, and fibrosis using H & E, Masson's Trichrome and Evans Van Gieson staining. RESULTS: Histology demonstrated cell types, local mesh reaction, and progressive evidence of solder reabsorption mimicking normal healing and bioincorporation. Mersilene groups demonstrated normal arrangement of collagen-rich layers around mesh. CONCLUSION: Collagen-based tissue soldering permits normal wound healing and may mitigate use of staples. Further development of this strategy is warranted.

Animals↗
PubMed1987

Effect of tissue processing on colloidal gold cytochemistry.

The aim of cytochemical techniques is to localize specific biochemical components in particular tissue and cell compartments. However, since preparation of tissues for structural observation results in major alterations of the properties of their components, a major problem is to retain an adequate degree of their biochemical properties as well as adequate structural preservation. In the present study, we describe results obtained using various colloidal gold cytochemical techniques on tissues processed through different approaches. We found that any manipulation of the tissue during its processing can result in modifications of tissue components, leading to problems in cytochemistry. Indeed, washing of the tissue before fixation, the nature of the fixative solution, the chemical basis of the resins, and the physical conditions of embedding can all introduce changes in tissue components which can be cytochemically demonstrated. This has been illustrated with application of the protein A-gold, lectin-gold, and enzyme-gold cytochemical techniques on tissues submitted to different processings: fixation by perfusion or by immersion; glutaraldehyde vs paraformaldehyde fixative solutions; cryo-ultramicrotomy; embedding in epoxy, GMA, Lowicryl, or LR resins. The results obtained have demonstrated that conditions for optimal labeling must be worked out for each class of binding sites, and that no single procedure can be recommended as THE best approach in cytochemistry.

Acrylic Resins↗
PubMedDate not supplied

The use of percutaneous sutures for graft fixation in rhinoplasty.

We reviewed our 10-year experience using percutaneous suturing to secure cartilage grafts in rhinoplasty. A total of 382 patients having up to 4 percutaneous sutures per surgery were analyzed. Cases using this suture technique included lower lateral onlay grafts, dorsal onlay grafts, tip grafts, and intact conchal cartilage grafts to repair nasal valve collapse. Sutures are ideally placed in a horizontal mattress fashion beginning at the skin surface, continuing into the nasal cavity, and then out through the skin. Sutures are strategically placed to secure the graft and at times stent open the nasal valve. The suture is tied loosely over the skin taking in to consideration the anticipated edema formation. There is no need to use a bolster technique. Sutures are removed at the second postoperative visit. Long-term follow-up reveals precise graft placement. There are in most instances no visible suture marks and our results achieve over 95% patient satisfaction. Percutaneous suture placement is a simple technique that allows precise graft placement and fixation without resultant scarring. It coapts the skin and soft tissues to the grafted nasal skeleton and prevents fluid accumulation, hematoma formation, and graft migration. We believe that it aids in decrease long-term tissue edema and excessive fibrosis, therefore promoting quicker healing. Our patients are both functionally and cosmetically pleased.

Cartilage↗
PubMed2004

Atomic force microscopy of BHK-21 cells: an investigation of cell fixation techniques.

The atomic force microscope (AFM) has been used to image a wide variety of biological samples, including cultured cells, in air. Whilst cultured cells have been prepared for AFM analysis using a variety of matrices and fixatives, a definitive study of sample preparation and its effects on cell morphology has not, as far as the authors are aware, previously been reported. Although a considerable number of cell fixatives exist, no single fixative is ideal for all investigations. Prior to the performance of specialised techniques, such as atomic force microscopy of cultured cells in air, the cell fixation method must be investigated and optimised. The fixative abilities of 2% paraformaldehyde-lysine-periodate, 0.25% glutaraldehyde, paraformaldehyde-glutaraldehyde, 4% phosphate-buffered formal saline, 1% formaldehyde, methanol:acetone, formal saline, 4% paraformaldehyde and ethanol:acetic acid were assessed in this study. A qualitative assessment system was used to evaluate the efficacy of the above fixatives using conventional fixation criteria (i.e. the presence of fibroblastic morphology consistent with optical microscopy and the absence of fixation artifacts). The optimal fixative was identified as 4% paraformaldehyde, which was capable of providing optically consistent images of BHK-21 (fibroblastic) cells, whose heights remained within the measurement capability of the AFM instrument used in this study.

Animals↗
PubMedDate not supplied

Subjective vs. objective analysis of the corneal endothelial cells in the rabbit cornea by scanning electron microscopy - a comparison of two different methods of corneal fixation.

OBJECTIVE: To illustrate a method of quantitative scanning electron microscopy (SEM) for the evaluation of the corneal endothelia of small animals by comparing two commonly used fixation methods. ANIMALS STUDIED: Female New Zealand white rabbits, aged 10-12 weeks. PROCEDURES: The corneas were either dissected from the eye and placed in fixative (2% glutaraldehyde in 80 mm cacodylate, pH 7.4) or the whole eyeball immersed in fixative solution and the cornea dissected later. Lower (x 200) and high magnification (x 1,000-5,000) images were inspected for overall appearance. Magnification images (x 500) were used to measure the areas of 100 cells, and cell density (CD) calculated from the average area. RESULTS: Both fixation protocols yielded an intact endothelial surface, but the dissect- then-fix protocol resulted in more creasing and distortion artifacts that were avoided with the whole-eyeball fixation. Overall, the CD values were higher if the dissect-then-fix method was used, and the uniformity of the cell mosaic was less. The median CD values for the central, mid-peripheral and peripheral regions following dissect-then-fix protocols were 7,693, 7,353, and 7,071 cells/mm(2) (average eight corneas at each location). If the whole-globe fixation was used, the median CD values were 6,098, 5,747, and 4,785 cells/mm(2). CONCLUSION: Cell density values in SEM can be very different according to the fixation method used. A distinct regional difference in CD was evident, which was more pronounced if the cornea was fixed prior to being dissected from the eye.

Animals↗
PubMedDate not supplied

Alcoholic carbowax prefixation and formal alcohol fixation. A new technique for urine cytology.

OBJECTIVE: To compare the cellular yield and sensitivity of cancer detection by urine cytology using alcoholic carbowax as a prefixative and formal alcohol as a final fixative with the conventional centrifugation-and-smearing method. STUDY DESIGN: Four hundred seventeen voided urine specimens were examined. Each specimen was divided into two equal portions. One portion was centrifuged and wet fixed (CS). The other was treated with alcoholic carbowax as a prefixative and then fixed in formal alcohol (CW). The smears were studied for cellular yield, morphology, cancer detection rate and typing. RESULTS: The CW method yielded significantly more cells with excellent morphology. It detected 25 more abnormal cases out of a total of 86, including 5 more transitional cell carcinomas, as compared to the CS method. This finding was statistically significant (P < .0000003). CONCLUSION: The CW method is simple and enhances cellular yield and the cancer detection rate as compared to the conventional centrifuge-and-smear technique.

Alcohols↗
PubMed1994

The preparation of native livers for morphological studies.

We describe experiences with a perfusion fixation apparatus that was used for studies on approximately 500 native livers. Immediately after excision of the specimen, small samples from needle biopsy specimens are obtained for snap-freezing and the remaining portions of the specimens are fixed and embedded in paraffin. In the laboratory, the portal vein and, if possible, the hepatic artery or common hepatic duct are then cannulated and the livers are perfused for 3 days with Kaiserling's solution. An electric pump drives the perfusion apparatus and allows the formalin to cascade through stacked plastic containers, with the specimens attached to the inflow nozzles for the fixative. Eight or more livers (or other organs and specimens) can be accommodated simultaneously. Angiograms or cholangiograms can be prepared before or after fixation; we prefer the latter. The livers are then sliced with an extra-long knife, which minimizes cutting marks. Most preparations are thoroughly fixed and yield excellent specimens, not only for routine microscopic study but also for special methods such as scanning electron microscopy and trace metal analysis. The liver slices can be stored indefinitely, which allows long-range collection for routine review or research purposes. In approximately 5% of the cases, specimens cannot be perfused properly and thus are unsuitable for this type of preparation. With autopsy specimens this percentage is higher, probably because of postmortem clotting. Gravity perfusion of the livers before placement into the apparatus generally enables identification of specimens with incomplete filling of the vasculature.

Angiography↗
PubMed1992

Sensitivity of the polymerase chain reaction for detecting human T-cell leukemia virus type I sequences in paraffin-embedded tissue. Effect of unbuffered formalin fixation.

Recently, the application of the polymerase chain reaction (PCR) to formalin-fixed paraffin-embedded tissue has been reported. But formalin, especially unbuffered formalin, is known to break DNA into small fragments. DNA extracted from MT-2 cells fixed in unbuffered formalin for various periods of time were subjected to the PCR and the effect of unbuffered formalin fixation on the ability of the PCR to detect exogenous sequences; i.e., human T-cell leukemia virus type I (HTLV-I) proviral DNA, was examined. The sensitivity of the PCR decreased as a function of both the duration of fixation and the length of the expected DNA products. When the expected length of the PCR product was about 200 bp, a slight decrease in the sensitivity was observed after 4-day fixation. When it was about 300 bp, a similar decrease was observed following 4-h fixation. In the case of a 500 bp product, the sensitivity began to decrease after 30-min fixation and a 100-fold decrease was observed after 10-day fixation. A decrease was not observed, however, with a 100 bp product. The appropriate design of primers, especially with regard to the length of the amplified product, is essential to keep the sensitivity of the PCR, particularly when the target tissues have been fixed in unbuffered formalin.

Base Sequence↗
PubMed1996

Techniques for reduction and posterior fixation through the anterior approach.

Pelvic fractures, especially those of a Malgaigne type, frequently occur in patients who are polytraumatized. Sacroiliac disruption reflects a massive, disruptive energy force with concurrent soft tissue injury. Fixation through an anterior approach facilitates the accurate and stable fixation and avoids many soft tissue wound problems associated with open anterior approaches. The absolute indication for the approach is a posterior disruption of the sacroiliac joint with or without an iliac wing fracture that is unreducible by closed reduction techniques.

Bone Plates↗
PubMed1992

Detection of proliferating cell nuclear antigen in diagnostic histopathology.

We describe the effects of tissue preservation, fixation time, and hydrolytic treatment on the detection of proliferating cell nuclear antigen (PCNA) by immunoperoxidase staining with three commercial anti-PCNA antibodies (19A2, 19F4, PC10). Our goal was to provide guidelines for PCNA immunohistochemistry in formalin-fixed, paraffin-embedded specimens. In proliferative cell compartments, nuclear staining was achieved with all three antibodies. In some cases PCNA was also expressed in non-proliferative, histologically normal tissues associated with tumors or other lesions elsewhere. In most autopsy specimens PNCA immunoreactivity was markedly diminished as compared with similar surgical specimens. Incubation overnight with primary antibody at 4 degrees C enhanced PCNA immunoreactivity over incubation at 42 degrees C for 45 min. Pre-treatment with 2 N HCl did not increase staining. Staining with the PC10 antibody was much better preserved than staining with the antibodies 19A2 and 19F4 after prolonged formalin fixation of surgical specimens and in tissues obtained at autopsy. With all three antibodies, however, PCNA immunoreactivity was well preserved during formalin fixation for 8-24 hr and during fixation delays for 8 hr at room temperature. This indicates that PCNA is stable under conditions routinely encountered in diagnostic surgical pathology and facilitates its potential use as a diagnostic proliferation marker.

Autoantigens↗
PubMed1989

Fixation and demineralization of bone tissue for immunohistochemical staining of neuropeptides.

The present study in the rat demonstrates the feasibility of applying immunohistochemical staining techniques on bone tissue for studies of substances such as neuropeptides contained in nerve fibers. Two fixation procedures, as well as the influence of demineralization on neuropeptide antigenicity, were studied in bone and for comparison in small intestine. In vivo perfusion with paraformaldehyde and picric acid, followed by demineralization in a solution of either EDTA-cacodylate or buffered EDTA-sucrose, proved to be the most appropriate with respect to preserved antigenicity. Antibodies to 23 neuronally related substances were tested. In the bone tissue, immunoreactivity was found to four neuropeptides: substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, and neuropeptide Y, and also to the catecholamine-synthesizing enzyme tyrosine hydroxylase. The described method for identifying intraosseal neuropeptides offers a new means of studying skeletal innervation and bioactive substances in bone tissue.

Animals↗
PubMed2000

A new interference nail fixation for anterior cruciate ligament soft tissue grafts.

We present a new device for femoral and tibial fixation in anterior cruciate ligament reconstruction using soft tissue. We evaluated the method biomechanically in pullout tests using human cadaver knee joints. Fixation strengths ranged from 300 +/- 60 to 600 +/- 50 N (mean 400 +/- 50 N). This corresponds to loads in the graft during aggressive rehabilitation although cadaveric specimens were used. With respect to primary fixation strength this implant is a reasonable alternative for anterior cruciate ligament graft fixation.

Aged↗