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PubMedDate not supplied

Cytochemical detection of endogenous peroxidase in the acinar cells of the hamster submandibular gland.

The presence of endogenous peroxidase activity in the hamster submandibular gland was investigated cytochemically by light and electron microscopy using diaminobenzidine methods. After fixation of tissue with 2% paraformaldehyde-2.5% glutaraldehyde and incubation in a DAB reaction medium containing 0.01% H2O2, the peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, secretory granules and the Golgi apparatus in both the acinar and granular duct cells of the submandibular gland. This is in contrast to earlier investigators who failed to detect peroxidase activity in acinar cells of the hamster submandibular gland and reported that peroxidase is localized only in the granular duct cells. The discrepancy may be caused by differences in experimental procedures. It is suggested that fixation of tissue with a high concentration of glutaraldehyde and incubation in a DAB reaction medium containing a high concentration of H2O2 inhibits the peroxidase activity of acinar cells in the hamster submandibular gland.

3,3'-Diaminobenzidine↗
PubMed2006

Soft tissue graft interference fit fixation: observations on graft insertion site healing and tunnel remodeling 2 years after ACL reconstruction in sheep.

Using soft tissue grafts for anterior cruciate ligament (ACL) reconstruction, insertion site healing plays a crucial role in the long-term fate of the graft. It has been shown in an experimental animal study that using a soft tissue graft and anatomic graft fixation, a direct ligamentous insertion alike the native ACL developed 24 weeks postoperatively. Yet there are no reports on the long-term insertion site healing of anatomically fixed soft tissue grafts. The objective of this study was to evaluate graft insertion site healing, the intra-tunnel fate of the graft and its osseous replacement 2 years after ACL reconstruction in sheep. The left ACLs of six sheep were replaced by an autologous flexor tendon split graft and anatomically fixed with biodegradable poly-(D, L-lactide) interference screws. Animals received polychromic sequential labeling at different points in time to determine bone apposition per period. For evaluation of the insertion site healing and intra-tunnel changes, MRI scans were taken in vivo. Following sacrifice, radiographic imaging, conventional histology and fluorescence microscopy was undertaken. Most of the specimens showed a wide direct ligamentous insertion. It showed patterns alike the direct ligament insertion seen in intact ACLs. The intra-tunnel part of the graft had completely lost its tendon-like structure and in two cases, it was separated from the graft insertion by a thick bony layer. The biodegradable interference screw was fully degraded in all specimens. Ossification of the former drill tunnels was intense, showing only partial-length tunnel remnants in one femoral and three tibial specimens. As the graft heals to the joint surface and the aperture site is closed with soft tissue, mechanical stress of the intra-tunnel part of the graft is eliminated and the bone tunnel is protected from synovial fluid, resulting in osseous bridging of the tunnel aperture site, accelerated intra-tunnel graft resorption and its osseous replacement.

Animals↗
PubMed1996

Ultrastructural localization of activity of phosphatases by low temperature incubation of unfixed cryostat sections.

In the present study, we demonstrate the activity of several phosphatases ultrastructurally in long-term (up to 24 months) cold-stored (-80 degrees C) rat tissues. Phosphatase activity was histochemically studied with the use of unfixed cryostat sections in combination with low temperature (4 degrees C) incubation conditions in order to prevent inactivation of enzyme activity and to limit the loss of ultrastructure. 5'-Nucleotidase activity was observed at plasma membranes, mainly at bile canalicular membranes of hepatocytes in liver. Thiamine pyrophosphatase activity was detected not only in trans side cisternae but also in medial and cis side cisternae of Golgi complexes in the parotid gland. Glucose-6-phosphatase activity was localized in endoplasmic reticulum as well as at the outer membrane of the nuclear envelope. Acid phosphatase reaction product was found in lysosomes. Furthermore, the localization patterns of 5'-nucleotidase and thiamine pyrophosphatase activity were compared with those obtained after different fixation procedures such as immediate chemical fixation of tissues or fixation of tissues after freezing and thawing. The results showed similar localization patterns of these enzymes after the different pretreatments. However, with respect to the ultrastructural morphology, some damage was observed in unfixed material after incubation. It can be concluded that the procedure described here enables ultrastructural localization of activity of phosphatases in long-term cold-stored tissues. This procedure will be useful for a retrospective study on archival material when histochemical parameters are needed.

5'-Nucleotidase↗
PubMed1989

Buffered phenol formaldehyde (pH 7.0 and pH 5.5): improved fixation in an enclosed tissue processor.

The addition of 2% phenol has a marked adjuvant effect on 4% formaldehyde as a fixative. Sequential use of phenol formaldehyde buffered to pH 7.0 and pH 5.5 results in improved fixation in material rapidly advanced to paraffin in an enclosed tissue processor. Sections stained with alum haematoxylin and eosin show improved nuclear and cytoplasmic detail, reduced tissue shrinkage and an absence of formalin pigment. Excellent results are obtained with conventional special stains and immunoperoxidase methods.

Fixatives↗
PubMed2001

Neurovascular risk of bicortical tibial drilling for screw and spiked washer fixation of soft-tissue anterior cruciate ligament graft.

PURPOSE: As the use of soft-tissue anterior cruciate ligament (ACL) grafts, including hamstring grafts, has become more prominent and the benefits of aggressive rehabilitation have become clear, maximizing fixation with screw and spiked washers is important. Bicortical fixation may be superior. We were concerned about potential neurovascular risks and designed this study to define the posterior neurovasculature structures at risk when drilling for bicortical tibial screw fixation during ACL reconstruction. Type of Study: Consecutive sample. METHODS: We placed the tibial tunnel arthroscopically in 10 cadaveric knees using a standard tibial drill guide. Accurate tibial tunnel position was documented in each knee by lateral radiograph. A 4.5-mm bicortical drill hole was placed perpendicular to the tibial surface 1 cm distal to the tibial tunnel. The distances from the posterior tibial drill exit point to nearby neurovascular structures were measured with a caliper. RESULTS: The closest structure to the exit point was the bifurcation of the popliteal artery/vein (11.4 +/- 0.6 mm; range, 8.4 to 14.0 mm). The next closest was the anterior tibial vein (11.7 +/- 1.6 mm; range, 3.5 to 22.8 mm). The closest any individual hole came to a neurovascular structure was 3.5 mm from the anterior tibial vein. CONCLUSIONS: Bicortical drilling for fixation of soft tissue grafts appears reasonably safe. The structures at greatest risk for injury are the bifurcation of the popliteal artery/vein and the anterior tibial vein.

Journal Article↗
PubMedDate not supplied

Determination of fixation strength of large-diameter interference screws.

Small-diameter interference screws are a widely used method of securing bone-tendon grafts during anterior cruciate ligament reconstructive procedures. Recent reports document tunnel widening and subsequent loss of fixation, leading to a need for alternative fixation methods. In cadaver tissue, the fixation strength of large-diameter (11-, 13-, and 15-mm) interference screws was tested and compared with that of clinically used 9-mm diameter screws. Patellar-tendon sections were fashioned into three grafts, each with bone plugs measuring 10 x 20 x 7.5 mm. Tunnels were created in the epiphyseal bone of the femurs and tibias, resulting in gap sizes of 4.5-, 6.5-, 8.5-, and 10.5-mm for the 9-, 11-, 13-, and 15-mm diameter screws, respectively. Grafts were pulled from the tunnel at a rate of 51 cm/min. Average fixation strengths were 276, 302, 328, and 328 N for the 9-, 11-, 13-, and 15-mm screws, respectively. No significant difference existed between the fixation strengths (ANOVA, P < or = .05).

Aged↗
PubMed2006

Diagnosis and quantification of bone marrow fibrosis are significantly biased by the pre-staining processing of bone marrow biopsies.

AIMS: Marrow fibrosis (MF) is an unfavourable, often lethal complication of haematological neoplasms. Although biopsy technique and staining procedure are standardized, the prestaining processing of bone marrow biopsies (BMBs) varies markedly without any existing data on its significance for the diagnosis of MF. METHODS AND RESULTS: In this study on 712 BMBs from 296 patients with chronic idiopathic myelofibrosis (CIMF), chronic myeloid leukaemia (CML), or healthy bone marrow, MF was a characteristic complication of CML and CIMF. However, diagnosis and quantification of MF and detection of its prognostic significance were significantly biased by fixation, decalcification, embedding, marrow tissue shrinkage during biopsy processing and the thickness of marrow sections (P < 0.000005). The relevance of these influences was explained by their effect on the marrow volume to which the fibre content was related, whereas the stainability of fibres was not affected. Semiquantitative grading of fibrosis and measurements of fibre density could not be adjusted to various methods of processing of bone marrow biopsies (P < 0.003). CONCLUSIONS: Evaluations of MF and its prognostic significance should consider the bias due to the prestaining processing of BMBs and the necessity of an adjustment to the thickness of tissue sections and the degree of marrow tissue shrinkage.

Adult↗
PubMed1992

Fixation for biological ultrastructure. II. Cross-linking of bovine serum albumin by nanosecond pulses of ionizing radiation.

Very large doses of ionizing radiation were delivered quickly to concentrated albumin solutions by pulses of 500 keV in air. The electrons penetrated the aluminium foil bottom of the test cell and into the solution. A dose of 4.6 Mrad (4.6 x 10(4) Gy) produced a gel in the albumin solution comparable to the long-term effect of 0.36% glutaraldehyde in the same albumin solution. The cross-links created by the radiation leading to gel formation are probably irreversible and quite different from those leading to gelation in the glutaraldehyde-albumin reaction. Single large pulses of ionizing radiation may be useful for fast fixation of cells and tissues for microscopy.

Equipment Design↗
PubMed1984

In vitro models reflecting the action of strong acids on integumental tissue.

The discoloration of white rat hair by acid preparations has been employed as a model reflecting local tissue destruction by intravital fixation as contrasted to tissue dissolution. Nitrate reduction products in equilibrium with nitrite ion in moderately strong acid result in much more rapid tissue fixation than is accomplished by acid preparations alone. Parallel differences were found in the color reaction of normal skin to different acid preparations. The rapid fixing capacity demonstrated by these models may be useful for sharply localized destruction of topical lesions clinically.

Animals↗
PubMed1996

Electron microscopic demonstration of intracelluar promethazine accumulation sites by a precipitation technique: application to the cerebellar cortex of the mouse.

A method is described that allows electron microscopic identification of the phenothiazine neuroleptic promethazine after supravital intracardiac injection of high drug concentrations (greater than or equal to 3 %). The cerebellar cortex of the mouse was used for the investigation. This procedure is based on simultaneous fixation of drug and tissue by immersion in a paraformaldehyde-glutaraldehyde solution with the addition of phosphomolybdic acid. The electron microscopic investigation revealed that the drug could easily be identified as an electron-dense precipitate. Subpopulations of neurons exhibited a higher affinity for the drug than others, but no preference for any nerve cell type was detected. Closer study showed that promethazine accumulated primarily within the cytoplasm, where it was bound to the endoplasmic reticulum. Furthermore, promethazine storage was observed within mitochondria and vesicular structures. Drug binding was also seen in the regions of synapses but without any predilection for these structures. Because this precipitation technique also appears to be well-suited for the ultrastructural identification of other drugs, it offers multiple possibilities for application.

Animals↗
PubMed2002

Continuous-specimen-flow, high-throughput, 1-hour tissue processing. A system for rapid diagnostic tissue preparation.

CONTEXT: Current conventional tissue-processing methods employ fixation of tissues with neutral buffered formalin, dehydration with alcohol, and clearing with xylene before paraffin impregnation. Because the time required for this procedure is usually 8 hours or longer, it is customary to process tissues in automated instruments throughout the night. Although this time-honored method continues to serve histology laboratories well, it has a number of shortcomings, such as a 1-day delay of diagnosis, the need to batch specimens, the relatively large volumes and toxicity of reagents used, and the extent of RNA degradation. OBJECTIVE: To describe a rapid new method of tissue processing using a continuous-throughput technique. Design.-We used a combination of common histologic reagents, excluding formalin and xylene, as well as microwave energy, to develop a rapid processing method. The effect of this method on the quality of histomorphology, histochemistry, immunohistochemistry, and RNA content of processed tissue was compared with that of adjacent tissue sections processed by the conventional processing technique. We also assessed the impact of this rapid processing system on our practice by comparing the turnaround times of surgical pathology reports before and after its implementation. RESULTS: The new processing method permitted preparation of paraffin blocks from fresh or prefixed tissue in about 1 hour. The procedure allowed continuous flow of specimens at 15-minute intervals. It eliminated the use of formalin and xylene in the processing and used considerably lower volumes of other chemical reagents. Histomorphologic, histochemical, and immunohistochemical results were comparable to the parallel sections prepared by the conventional method. The new technique, however, preserved higher quality RNA. Use of the new methodology led to the diagnosis and reporting of more than one third of surgical pathology specimens on the same day that they were received, as compared to 1% of same-day reporting before the implementation of the rapid processing system. CONCLUSION: The quality of hematoxylin-eosin, histochemical, and immunohistochemical tissue sections provided by the new system is comparable to that obtained following the conventional processing method. The new system preserves RNA better than the conventional method. It also shortens the processing time to about 1 hour from the receipt of fresh or prefixed tissue, eliminates the need for formalin and xylene, and reduces the volume of other chemicals. Most importantly, it impacts overall patient management by allowing for considerably shorter turnaround times for completion of surgical pathology reports.

Female↗
PubMed2002

[Immunochemistry evaluation of HER2 status in infiltration breast cancer: technical protocol and interpretation guidelines].

In Europe, patients who may benefit from Herceptin((R)) (an HER2 targeted drug) are currently selected by immunohistochemistry (IHC). Reliable detection of HER2 status is essential to the appropriate usage of Herceptin(R), because its specificity is limited to tumours overexpressing HER2. It is essential that the IHC evaluation of the HER2 status of a mammary carcinoma be optimized and reliable. This technical paper reviews the different steps of the IHC technique, the controls and, the rules for interpretation. The sensitivity of the IHC technique must be adjusted so as not to produce false negatives or false positives. As opposed to other methods, it can be carried out whatever the fixation conditions of the tissues. The interpretation of the immunostains also requires training; it is fraught with problems for intermediate positivities. The ideal score to evaluate HER2 status has not yet been defined. It will thus be necessary to report the percentage of stained cells, the intensity of the staining, and, in respect to Herceptin((R)) treatment, the HercepTest scoring system (recommended in the package insert). Once acquired, this knowledge must be perpetuated by the observation of rules of good technical practice (internal and external controls, quality assurance programs). FISH should be used for complementary assessment of 2+ cases (on condition that they have not been fixed in Bouin's liquid) and for the calibration of the IHC technique.

Breast Neoplasms↗
PubMed1996

[Experimental studies of segmental bone defects treated by skeletal external fixation].

Bony tissue of an average of 13.6% of the original length (OL) was resected from the tibia shaft in 26 mature goats. The fracture ends were compressively fixed with skeletal external fixation (SEF). Simultaneously, upper metaphysiotomy was performed to lengthen the tibia with a lengthening rate of 1.0 mm/d and a total lengthening 20.14% of OL. The restoration of blood circulation and the hemodynamic changes during the repair process of the compressively fixed bone ends and the metaphysiotomy site were studied with angiography, microvascular cast, Chinese ink permeated transparent thick slide, and radioactive microsphere measurement of regional blood flow (RBF). It was found that there were abundant vascular networks in the lengthened zone. The blood vessels not only could keep up with the same lengthening rate of growing, but also were gradually converted into the Haversian system in the newly formed cortex of the lengthened area. In the 2nd week after the cessation of bone lengthening, the RBF of the lengthened zone reached the maximum (54.56 +/- 8.53 ml/mm/100 g). It was also found that in the 7th postoperative week, the vessels of the cortex of the osteotomy shortening ends could grow across the space between them and anastomose with each other. The RBF at this time was 31.82 +/- 2.85 ml/mm/100 g. In the 12th week after operation, the RBF was still 2 times higher than that of the normal cortex (3.02 +/- 0.13 ml/mm/100 g). The primary healing could be observed under the wellreduction and stable compressive external fixation. These findings provide a theoretical basis for the clinical application of SEF for the treatment of tibial shaft defects or nonunion associated with limb shortening.

Animals↗
PubMed2001

Improved scalp surgery results by controlling tension vector forces in the tissues by galea to pericranium fixation sutures.

BACKGROUND: Tension, manifesting at the time of wound closure, limits the extent of tissue excision and predisposes to complications. Extensive undermining may be an unreliable technique to reduce tissue tension in surgery. Pericranium is a rarely used tissue in scalp surgery. OBJECTIVE: To show that the surgical technique of deep plane fixation reduces tissue tension at and adjacent to the wound, allowing relatively greater excision and reduced tension closure, and extensive undermining may be an ineffectual and potentially harmful surgical technique. I also update the surgical anatomy of the pericranium and determine its utility in scalp reconstructive surgery. METHODS: More than 1000 scalp operations, including 700 clinical investigative procedures, were done which included pericranial flaps, subgaleal and subperiosteal scalp reductions, and deep plane fixation. RESULTS: The alopecia removal operation described herein utilizing deep plane fixation is largely complication free and achieves tension-reduced closure and between 18% and 50% greater tissue removal compared with conventional alopecia reduction procedures. CONCLUSION: Pericranium is a valuable and readily usable tissue in reconstructive scalp surgery.

Alopecia↗
PubMed1986

A technique for the electron microscopic autoradiography of Al adenosine receptors in brain tissue. Ligand-receptor fixation by osmium tetroxide.

A procedure for the electron microscopic autoradiography of Al adenosine receptors is described. Fresh tissue slices from rat hippocampus were incubated with the radioactive adenosine analogs: Cyclohexyl[3H]adenosine, 5'-N-ethylcarboxamido[3H]adenosine or or [125I]-iodohydroxyphenylisopropyladenosine. Various fixation agents were tested with respect to the retention of these ligands by the tissue. While most of the ligands were lost in aldehyde fixation they were retained by osmium tetroxide probably via a crosslinking reaction. The final method of choice was an aldehyde prefixation (in the case of [125I]-iodohydroxyphenylisopropyladenosine with 4% buffered paraformaldehyde) during which more than 90% of the nonspecifically bound ligands were washed out while 40% of the specifically bound ligands remained. Subsequent fixation with osmium tetroxide (1%) allowed a standard protocoll for dehydration and embedding to be used with only minimal (less than 5%) further loss of the ligands. Electron microscopic autoradiography provided evidence for a specific distribution of the binding sites for [125I]-iodohydroxyphenylisopropyladenosine.

Animals↗
PubMed1993

Distinction between artefactually shrunken and truly degenerated 'dark' neurons by in situ fixation with microwave irradiation.

Dark, shrunken neurons frequently occur as artefacts in immersion fixed tissue. Perfusion fixation will prevent artefacts of this type. However, morphologically identical neurons have been described as truly degenerated cells in perfusion fixed brains in various pathological conditions. Since adequate perfusion is difficult to obtain in some pathological conditions, the question still remains whether the dark neurons found in some of these situations are true in vivo phenomena or artefacts caused by inadequate fixation. In the present study rat brains with cryogenic lesions were fixed in situ by microwave irradiation. With this method no artefactually changed dark neurons were observed in the normal parts of the brains. In the cryogenic lesions, however, a narrow rim of dark, shrunken neurons occurred adjacent to the normal cortex. This zone was identical to that observed in perfusion fixed tissue. Since inadequate fixation due to uneven perfusion of the damaged tissue is prevented with this method, we suggest that the neuronal changes represent true in vivo phenomena. Fixation with microwave irradiation can thus be used to differentiate between artefactually changed and truly degenerated dark neurons in various pathological conditions.

Animals↗
PubMed1984

Restoration of antigenicity of tissue antigens, cell-bound immunoglobulins and immune deposits in paraffin-embedded tissue. The influence of fixation and proteolytic enzymatic digestion.

The possibility of preservation and restoration of antigenicity of some antigens in paraffin-embedded tissue was evaluated by direct immunofluorescent technique on deparaffinized sections. Fixation with 96% ethanol-1% acetic acid, 10% neutral buffered formalin and p-formaldehyde was useful for the preservation of tissue antigens and immune deposits, whose antigenicity could be easily restored by trypsin digestion. Neutral buffered formalin was also a satisfactory fixative in immunofluorescent staining on lymphocyte/plasma cell-bound immunoglobulins. Fixation with alcohol-Bouin's fluid showed contrast results; feasible for staining of cell-bound immunoglobulins, but poor for that of glomerular immune deposits. After papain digestion, BSA and lysozyme, antigens of immune complexes, were easily detected in experimental chronic serum sickness glomerulonephritis. Pepsin was more efficient than trypsin in restoring the antigenicity of renal tissue antigens such as fibronectin and polyantigenic basement membrane, but the brush border antigen of the proximal renal tubules was frail to the pepsin digestion. In general, the enzymatic digestion time necessary for the restoration of antigenicity was in parallel with fixation time. Results obtained have shown that deparaffinized sections could be used as satisfactory substrate for immunohistochemistry when proper fixation and efficient proteolytic enzymatic pretreatments were performed.

Animals↗