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PubMed1987

The effects of various fixatives on subsequent lectin binding to tissue sections.

The effects of ten fixation protocols on the subsequent binding of eight lectins to various mouse tissue sites have been systematically evaluated. The fixatives used were neutral and buffered formalin-saline, Bouin's fluid, 95% ethanol, Carnoy's fluid, calcium acetate-paraformaldehyde, and mercuric chloride both before and after removal of mercury pigment. These were compared with frozen sections of unfixed tissue and frozen sections post fixed in paraformaldehyde. Lectins used were PNA, DBA, SBA, BPA, UEA 1, GS I, GS II and MPA. Ethanol was found to be the superior fixative, closely followed by mercuric chloride. Paraformaldehyde was a poor fixative of both paraffin and frozen sections. It is recommended that, where a choice is possible, the fixation protocol appropriate to the particular lectin and tissue binding site is selected. Within certain limitations, formalin-saline proved an adequate fixative for the study of routine paraffin-processed tissue sections.

Animals↗
PubMed1988

A simplified plastic embedding and immunohistologic technique for immunophenotypic analysis of human hematopoietic and lymphoid tissues.

Routine fixation and paraffin embedding destroys many hematopoietic and lymphoid differentiation antigens detected by flow cytometry or frozen section immunohistochemistry. On the other hand, morphologic evaluation is difficult in flow cytometric or frozen section studies. A simplified three-step plastic embedding system using acetone-fixed tissues embedded in glycol-methacrylate (GMA) resin has been found to provide both excellent morphologic and antigenic preservation. With our system, a wide variety of antigens are detected in plastic sections without trypsinization or prolonged embedding procedures; pan-B (CD19, CD22), pan-T (CD7, CD5, CD3, CD2), T-subset (CD4, CD8, CD1, CD25) markers as well as surface immunoglobulin and markers for myeloid and mononuclear-phagocyte cells are preserved. In summary, modifications of plastic embedding techniques used in this study simplify the procedure, apparently achieve excellent antigenic preservation, and facilitate evaluation of morphologic details in relation to immunocytochemical markers.

Antigens, Surface↗
PubMed1988

Alkaline and acid phosphatase demonstration in human bone and cartilage: effects of fixation interval and methacrylate embedments.

Human bone and cartilage specimens were evaluated for acid and alkaline phosphatase localization following varying fixation periods for fresh or frozen tissue. Formalin fixations of up to 183 hr were followed by embedment in methyl methacrylate; frozen tissue was examined either without fixation or following fixation for up to 1 hr and subsequent glycol or methyl methacrylate embedding. The humeral epiphysis of a young patient with osteogenic sarcoma showed optimum acid and alkaline phosphatase localization following fixation for periods up to 15 hr and embedding in methyl methacrylate. Frozen costochondral junction from a newborn with osteogenesis imperfecta type II showed optimum acid and alkaline phosphatase localization following 30 min fixation in formalin and embedding in methyl methacrylate or after 5 min fixation and embedding in glycol methacrylate.

Acid Phosphatase↗
PubMed1986

The detection of endothelial cell antigens in cutaneous tissue using methacarn and periodate lysine paraformaldehyde fixation.

The use of monoclonal antibodies with endothelial cell specificity has prompted a search for methods of fixation which combine the morphology of paraffin-embedded tissue, with preservation of labile membrane antigens. Immunohistochemical staining using a variety of endothelial cell markers was compared in tissue fixed in formalin, methacarn, periodate lysine paraformaldehyde (PLP) and in frozen tissue. Whilst lectin-binding with Ulex europaeus agglutinin I (UEA) and localisation of Factor VIII-related antigen (FVIII RA) and laminin was well-visualised in methacarn-fixed and PLP-fixed tissue, fixation in PLP was necessary for the two monoclonal antibodies, PAL-E and EN4. PLP fixation has considerable potential for investigating the histogenesis of vascular tumours, particularly in Kaposi's sarcoma where frozen tissue represents a biological hazard. The normal staining pattern of human dermal vasculature is described in relation to the above endothelial cell antigens.

Acetates↗
PubMed1973

Observations on diseased pigs with high sulfate intake and normal tissue copper levels.

Disease in a large pig herd reared intensively and kept on sulfate-rich drinking water is described. It is the first report of diseased progeny of sows with high sulfate intake. Results of two surveys are presented, one for water with sulfate in excess of 2000 ppm and one for water with less than 1000 ppm. The management practices are described in detail. Disease of Survey I was manifested by high morbidity and mortality (50% of 600) in piglets, incoordination in piglets and some adult stock and osteopathy in piglets and weaners. In Survey II disease was less severe and restricted to piglets. Detailed histopathological studies revealed myelin deficiency in brain and spinal cord of sows and piglets, interferred endochondreal ossification of long bones of piglets and weaners, fatty changes of livers and interstitial nephritis in piglets and weaners. The changes in the nervous tissue were considered due to delayed fixation as tissue was only immersed in fixative and not perfused with it immediately after death. Similar changes have been described for pigs deficient in copper. Copper content of tissue and body fluids of pigs of this study were normal, as were the serum inorganic phosphate and total calcium levels. The bone changes observed have also been reported for rats given dextran sulfate injections, for pigs on experimental low-copper sulfate-enriched diet and for pigs reported low in copper and fed a diet supplemented with sulfide. The cause of the locomotor disturbance and mortality in piglets was not established.

Administration, Oral↗
PubMed1996

Microwave pretreatment improves RNA-ISH in various formalin-fixed tissues using a uniform protocol.

RNA is situ hybridization technique (RNA-ISH) is hampered by formalin fixation of tissues. This necessitates a pretreatment step of enzymatic digestion. However, to achieve the best results, digestion times and concentrations of enzymes have to be adapted for every specimen in a cumbersome procedure. Microwave pretreatment (MP) of formalin-fixed tissues has developed to become a powerful tool in immunohistochemistry (IHC) in recent years. To evaluate whether MP could also be helpful for RNA-ISH, we compared MP and conventional enzymatic pretreatment systematically using different tissues and various fixation times. As a model for RNA-ISH we chose the detection of kappa/lambda light chain mRNA in tonsils and lymph nodes with follicular hyperplasia and lymph nodes of patients with lymphocyte predominant Hodgkin's Disease (LPHD). Signal intensity obtained after MP was at least as good or dramatically enhanced as that obtained with optimized, single case adapted conventional pretreatment, the morphology being much better preserved after MP. These results confirm MP as a suitable method to unify the staining protocols in RNA-ISH, regardless of the duration of formalin fixation. Based on our results we recommend MP as a reliable and inexpensive method to enhance, standardized and simplify RNA-ISH.

Formaldehyde↗
PubMed1996

A comparative study of histological conditions suitable for both immunofluorescence and in situ hybridization in the detection of Herpesvirus and its antigens in chicken tissues.

Our objective was to identify an optimal single set of conditions for use in both indirect immunofluorescence assays (IFA) and in situ hybridization (ISH) to detect viral proteins and nucleic acids in avian lymphoid and neural tissues. Various fixatives were evaluated for use with IFA to detect turkey Herpesvirus (HVT) glycoprotein B (gB) and ISH to identify HVT mRNA in chicken tissues. A precipitating fixative (acetone) was compared to crosslinking fixatives [buffered glutaraldehyde-picric acid (BGPA), 10% formalin, and 4% paraformaldehyde] for both IFA and ISH using spleen, thymus, bursa, sciatic plexus, and brachial plexus of 28-day-old chickens. Four percent paraformaldehyde was found to be the optimal fixative for preservation of all chicken tissues examined with both IFA and ISH. Glass slide preparation, incubation temperatures, and tissue processing were each individually evaluated for ISH and IFA. Silylated slides provided the best retention of tissue sections for both procedures. For IFA, 37 degrees C was the ideal incubation temperature tested, whereas the optimal incubation temperature tested for ISH was 47 degrees C. Of the blocking agents compared, Evans blue dye prevented background fluorescence to a greater extent than either calf serum or bovine serum albumin. These findings provide a technical basis for investigations into various aspects of the molecular pathology of avian diseases.

Animals↗
PubMed1983

Soft-tissue reconstruction of the leg associated with the use of the Hoffmann external fixator.

Soft-tissue injuries and defects associated with severe fractures of the tibia treated with the Hoffmann external fixator were reviewed. Of 84 cases of open tibia fractures (classified as Gustilo's Type II and III), 54 patients with 55 injured limbs required soft-tissue reconstruction other than skin grafts or small rotational flaps. These 55 cases were constructed with 37 local musculocutaneous flaps (20 gastrocnemius, 17 soleus and other smaller muscle flaps), 15 free musculocutaneous flaps (14 latissimus dorsi, and one tensor fascia lata), and three medial gastrocnemius cross leg flaps. In all cases the soft-tissue reconstruction was done with the external fixator in place. All but three of the musculocutaneous flaps healed with no problems (94%). Bony healing was found in 30 cases (69.09%) at 18-month followup. There was one amputation after two failed local flaps and three late amputations due to patient's request (infected nonunion). There was a nonunion rate of 25.45% (14 cases) at 18 months postinjury. The Hoffmann external fixator has allowed bony stabilization and has not precluded the subsequent soft-tissue reconstruction or bone grafting. Muscle and musculocutaneous flaps, both local and free, have been employed in the reconstruction of the leg with minimal interference from the Hoffmann device.

Adolescent↗
PubMed1998

Growth factor stimulation of bone healing. Effects on osteoblasts, osteomies, and implants fixation.

Bone tissue has been shown to contain numerous cell-to-cell signalling peptides called growth factors. These growth factors are thought to have important regulating effects for bone remodeling and bone healing, due to their potent effects on bone cell metabolism. In vivo studies over the last half decade have demonstrated that growth factors candidates for future clinical use in orthopedic surgery. In numerous clinical situations enhanced bone formation and bone healing could lead to improved results of surgical procedures. This thesis describes the most important bone growth factors and their actions in vitro and in vivo. In vitro investigations of growth factor effects on osteoblast chemotaxis and metabolism are described as well as in vivo studies with growth factor stimulation of fracture healing and bone healing to prosthetic-like implants. In vitro results: Several growth factors exhibited chemotactic effects towards human osteoblasts. TGF-beta 1 and PDGF-BB had the strongest chemotactic effects, whereas PDGF-AA, IGF-1, and IGF-2 had less but significant chemotactic effects towards human osteoblasts. TGF-beta 1 exhibited the highest chemotactic potency with maximal activity at 100 pg/mL, whereas the other growth factors had maximal effects at 10-100 ng/mL. BMP-2 was found to have chemotactic effects toward human osteoblasts, human bone marrow osteoprogenitor cells, and U2-OS osteosarcoma cells. BMP-4 and BMP-6 were without any chemotactic effects towards these celltypes. Human bone marrow osteoprogenitor cells were the most responsive celltype to BMP-2 stimulation. Growth factor combinations resulted in synergic stimulative effects on different metabolic functions on human osteoblasts. Combinations with TGF-beta 1 and PDGF-BB strongly stimulated proliferation and chemotaxis. Combinations with TGF-beta 1, PDGF-BB and BMP-2 strongly stimulated an osteoblast differentiation parameter (alkaline phosphatase activity). The different growth factor combinations had no effect on collagen synthesis in human osteoblasts. In vivo results: Continuous application of 1 and 10 micrograms natural TGF-beta to a plated tibial osteotomy in rabbits increased mechanical bending strength and callus formation at 6 weeks observation. Diaphyseal cortical bone remodeling was not affected by the local growth factor application. In a dog model with unloaded implants surrounded by a gap, 0.3 microgram rhTGF-beta 1 adsorbed to gritblasted tricalcium phosphate coated implants, was able to enhance mechanical fixation, bone ingrowth and gap bone formation. 3.0 micrograms rhTGF-beta 1 had less but significant stimulative effect. In a weight-loaded model, 0.3 microgram rhTGF-beta 1, adsorbed to gritblasted tricalcium phosphate coated implants, was able to enhance bone ingrowth, without enhancement of mechanical fixation. In the unloaded model, 0.3 microgram rhTGF-beta 1, adsorbed to gritblasted hydroxyapatite coated implants, was able to enhance bone ingrowth, without enhancement of mechanical fixation. 3.0 micrograms rhTGF-beta 1 had no stimulative effects. The establishment of a biological implant fixation concept with growth factor absorbed to ceramic coatings of implants was successful. These data are promising for a possible future clinical usage of growth factors, especially for enhancement of bone healing to cementless prosthetic components.

Animals↗
PubMed1988

[Treatment of the open tibial fracture with the fixateur externe with special reference to osteosynthesis of the fibula].

In the treatment of open fractures of the lower limb and of fractures combined with severe damage of soft tissues, external fixation is favoured. Additional soft tissue damages in the compartments of the lower limb are avoided by using an external fixation in form of a clamp. In defect fractures, shattered fractures and in all fractures in the distal third the stability of clamp fixation is enhanced by plating of the fibula. Furthermore by osteosynthesis the reconstruction of the correct length of the lower limb can be performed. 36 fractures healed definitively in an average of 17.1 weeks by this procedure. Secondary procedures were: 8 cases with decompression of compartments, 11 cancellous bone grafts, 13 fixateur externes were removed because of the infection of the Schanz'screws, and healing was achieved by using a plaster cast. The therapeutical procedure was changed in 5 patients to a plate osteosynthesis and in 8 patients to an intramedullary nail.

Adolescent↗
PubMed2004

Pitfalls in diagnostic immunohistology.

The integration of immunostaining as an often-essential component of morphologic assessment makes it necessary that pathologists are familiar with the many technical and interpretive pitfalls that may arise because of the wide variety of factors that can significantly influence the ability to demonstrate relevant antigens in paraffin-embedded tissue sections. Pre-analytical variables that affect immunostaining include fixation, type of fixative, duration, temperature, and pH of fixation, variables in tissue processing, antigen loss resulting from delays in fixation, tissue necrosis, and levels of antigen expression. Analytical factors relate to the complex and sometimes capricious immunolabeling procedure that has as variables the specificity and sensitivity of the antibody clone, reagent dilution, detection system, and chromogen, and importantly, the method of antigen retrieval, which has its own set of variables such as time, temperature, method of heat generation, retrieval solution pH and molarity, and the synergistic action of proteolytic digestion. To obtain the highest diagnostic yield from immunostaining the correct questions must be asked and the pathologist must have familiarity with the characteristics of the antibody, its cross reactivity, if any, and localization of antigen in the cell to understand thresholds and cut-off levels and to recognize false-positive staining. Proper utilization of immunostaining requires that it is employed as a morphology-based technique and not interpreted in isolation.

Artifacts↗
PubMed2006

Graft laceration and pullout strength of soft-tissue anterior cruciate ligament reconstruction: in vitro study comparing titanium, poly-d,l-lactide, and poly-d,l-lactide-tricalcium phosphate screws.

PURPOSE: The aim of part 1 of this study was to compare the structural properties of tibia-graft-interference screw constructs of soft-tissue graft fixation via the anterior cruciate ligament (ACL) by use of different interference screws. In part 2 the influence of graft laceration as a result of 1 single interference screw insertion on the mechanical properties of the graft was evaluated. METHODS: In part 1 of the study, in a bovine knee model, soft-tissue graft fixation with titanium, biodegradable poly-D,L-lactide (PLDLA), and PLDLA-tricalcium phosphate (TCP) interference screws was performed, and the structural properties were determined by use of a materials testing machine at a rate of 12.5 mm/s. In part 2 the bone was cut after 1 single insertion of titanium, PLDLA, or PLDLA-TCP interference screws, and the mechanical properties of the graft were determined. RESULTS: Soft-tissue graft fixation with a biodegradable PLDLA interference screw showed a statistically significantly higher maximum load when compared with PLDLA-TCP and titanium interference screws. PLDLA-TCP screw fixation revealed a significantly higher ultimate load than titanium screw fixation. The mechanical properties of the grafts after 1 single insertion of PLDLA-TCP interference screws showed a statistically significantly higher stiffness, yield load, and maximum load when compared with the PLDLA and titanium interference screw groups. CONCLUSIONS: The results of this study suggest that the use of a titanium screw to fix a soft-tissue graft in cruciate ligament reconstruction surgery may cause damage to the graft. Soft-tissue ACL reconstruction fixed with a PLDLA interference screw resulted in a significantly higher pullout strength than ACL reconstructions fixed with a PLDLA-TCP or titanium interference screw. CLINICAL RELEVANCE: In contrast to fixation with a biodegradable interference screw, fixation with a titanium interference screw may damage the integrity of a soft-tissue graft in cruciate ligament reconstruction and should therefore be avoided.

Absorbable Implants↗
PubMed1983

Post mortem, ultrastructural cardiac muscle changes and anthracycline toxicity.

Anthracycline induced cardiomyopathy is commonly evaluated using the ultrastructural investigation of myocardial tissue. This is based to a large degree on subjectively evaluated, studies of post-mortem material which report various fin structural changes of cardiac tissue as being characteristic of this cardiomyopathy. We have studied the morphological variations of normal myocardial tissue in rats following perfusion fixation and immersion fixation of tissue obtained at death as well as at time points up to 6 hours after death, to examine the fine structural changes associated with immersion fixation and post mortal artefacts. Mitochondria profile area (reflecting mitochondrial swelling--a factor constantly reported as typical of anthracycline damage) increased significantly (p less than 0,01) if there was any delay between cell death and fixation. Many other fine structural changes were noted. We conclude that studies of this nature should ideally be carried out in experimental systems using perfusion fixation. Fixation procedures must always be defined in detail. If post mortem material must be used adequate information on the duration of the autolytic period must be given.

Animals↗
PubMed1995

Microwave fixation: understanding the variables to achieve rapid reproducible results.

The use of microwave irradiation for rapid chemical fixation of tissues in electron microscopy is a subject of current interest. The effects of water load size and location, sample placement in the oven cavity (hot or cold spots), and time on tissue preservation were examined. The use of a microwave container (4 dram vial) encased in 60 ml of ice in a 100 ml polyethylene beaker and a 0% power setting between two 100% power settings (time interval) provided reliable control of temperature during microwave irradiation. High brightness neon lights provided a quick and easy method to identify and map hot and cold spots within the oven cavity. Using microwave irradiation for rapid glutaraldehyde and osmium tetroxide fixation of tissues (Pacific yew needle and mouse kidney and liver) for electron microscopy yielded preservation equal or better than routine immersion fixation when a time interval, a cold spot (as the sample location), and an ice-encased vial were used during microwave fixation. These adaptations provided reliable control of fixation conditions in an 800 watt laboratory microwave oven.

Animals↗
PubMed1991

[Detection of mRNA by in situ hybridization in tissues fixed in Bouin's fixative and embedded in paraffin].

Human skeletal tissues fixed in Bouin's solution and embedded in paraffin were studied in order to identify cells responsible for production of types I and II collagens by in situ hybridization. The probes used were a double stranded cDNA fragment (type I collagen) and a synthetic oligonucleotide (type II collagen). The specificity of these probes labeled with 32P was proven in hybridizations to sections of human fetal femoral heads: fibroblasts and chondrocytes, known to produce respectively type I and type II collagen were only recognized by the corresponding probes. These results suggest that paraffin blocks available in pathology departments could be reexamined by in situ hybridization. Furthermore, the use of synthetic oligonucleotides without cloning the gene of interest could increase the usefulness of the technique in studies on human cartilage diseases and possibly on other diseases.

Acetates↗
PubMed2000

Adverse tissue reactions to bioabsorbable fixation devices.

Among 2528 patients operated on using pins, rods, bolts, and screws made of polyglycolic acid or polylactic acid, 108 (4.3%) were affected by a clinically significant local inflammatory, sterile tissue reaction. The three most common indications for the use of these fixation devices were a displaced malleolar fracture, a chevron osteotomy for hallux valgus, and a displaced fracture of the radial head. In 107 patients, the reaction was elicited by a polyglycolic acid implant, and in one patient by a polylactic acid implant. The incidences were 5.3% (107 of 2037) and 0.2% (one of 491), respectively. The adverse tissue responses to polyglycolic acid were seen 11 weeks after the operation, on average, whereas the reaction to polylactic acid occurred 4.3 years after fixation of an ankle fracture. The mild reactions consisted of a painful erythematous papule of a few weeks' duration. Those of medium severity had a sinus that discharged remnants of the implant for up to 6 months. In the patients affected by severe reactions, extensive osteolytic lesions developed at the implant tracks. The histopathologic picture was that of a nonspecific foreign body reaction. In four patients with vigorous reactions, an arthrodesis of the wrist or ankle later was necessary because of severe osteoarthritis. Several markers of increased risk of the occurrence of a foreign body reaction were found. These included a poorly vascularized bone section such as scaphoid, use of a quinone dye as an additive in the polymer, and an implant geometry with large surface area (screw versus pin or rod). For polyglycolic acid implants, the risk of an adverse tissue response in a given clinical situation can be estimated from the findings of this study. For slow degrading polymers like polylactic acid, however, the ultimate biocompatibility still is unsettled, and additional clinical research with long followup is required.

Absorbable Implants↗