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At least 451 records · Page 25Linked to original sources
PubMed2006

Tissue shrinkage after fixation with formalin injection of prostatectomy specimens.

Prostate cancer volume correlates with stage, grade, and progression after prostatectomy. When tumor volume is measured planimetrically, results are multiplied by a correction factor to compensate for tissue shrinkage caused by processing. Injection of formalin into prostatectomy specimens was suggested for improved fixation. Our aim was to investigate how this affects the prostate volume. We studied 142 radical prostatectomy specimens. All prostates were immersed in 10% formalin. In 84 prostates (59%) we also injected 20 ml of formalin before routine fixation. The prostates were weighed unfixed after injection and after final fixation. The specimens were sliced and totally embedded. The transverse diameters of the prostates were measured on unfixed specimens and microscopic sections. The average weight loss after final fixation was 5.8 and 8.6% for formalin-injected specimens and standard-fixed specimens, respectively (p<0.001). However, when total shrinkage was estimated from the transverse diameters, there was no difference related to fixation technique (p=0.59). The average linear shrinkage was 4.5%, corresponding to a volume correction factor of 1.15. We conclude that formalin injection for fixation of prostate tissue does not influence tumor volume calculation compared to conventional fixation.

Artifacts↗
PubMed1998

Collagen type IX and developmentally regulated swelling of the avian primary corneal stroma.

A critical event in avian corneal development occurs when the acellular primary stroma swells and becomes populated by mesenchymal cells that migrate from the periphery. These cells then deposit the mature stromal matrix that exhibits the unique features necessary for corneal function. Our previous work correlated the disappearance of collagen type IX immunoreactivity at stage 27 (5 1/2-6 days) with matrix swelling and invasion. To investigate further the mechanism of this disappearance, we employed immunohistochemistry after tissue fixation with Histochoice, a non-crosslinking fixative, immunoblot analysis of protein extracts, and gel substrate chromatography (zymography) to detect endogenous proteolytic activity. We found that corneas fixed in Histochoice retain immunoreactivity for type IX collagen for 1-2 days after corneal swelling. This immunoreactivity, however, becomes extractable from tissue sections of unfixed corneas at the time of initiation of stromal swelling and mesenchymal cell invasion. Immunoblot analysis confirmed that, following swelling, immunoreactivity for collagen IX decreased substantially in corneas, but not in the vitreous body, which served as a comparison. Analysis of ammonium sulfate (AS) fractions of such extracts indicated that, at the time of swelling, much of the immunoreactivity for type IX collagen in cornea shifted from the AS precipitate (containing high molecular weight molecules) to the AS supernatant (containing smaller fragments). In contrast, collagen IX immunoreactivity from the vitreous was precipitated by ammonium sulfate throughout the period of study. Collagen type II, a major fibrillar collagen in both the corneal stroma and vitreous, remained in the high molecular weight fraction at all times examined. Zymography detected the presence of the latent (proenzyme) form of gelatinase A (MMP-2) before corneal swelling and invasion (4 days), and both the latent and active forms of the enzyme after corneal swelling. This suggests tissue-specific, developmentally regulated proteolysis of collagen IX as a trigger for corneal matrix swelling.

Animals↗
PubMedDate not supplied

Simple technique to facilitate tissue sampling for electron microscopy.

The need to examine certain components of a specimen with the electron microscope is frequently recognized only after study of the paraffin sections, by which time the total specimen has usually been fixed in Formalin. Such specimens are frequently large, with the result that representative samples for electron microscopy may be difficult to isolate. A simple, quick, and inexpensive method for overcoming this sampling problem is described. This technique is best employed in laboratories that routinely use a single tissue fixative suitable for both light and electron microscopy. The specimen is taken direct from the Formalin, and very thin slices are prepared by hand with a blade, stained with methylene blue, and examined in the wet state under the microscope. On identification, representative tissue is removed by micro-dissection and processed further for electron microscopy.

Histological Techniques↗
PubMed2002

Histone mRNA in situ hybridization in assessing proliferative activity of normal and malignant cells.

Proliferative capacity is an important determinant of tumour biological behaviour. For research and diagnostic purposes, immunohistochemical techniques are usually applied in the assessment of tissue proliferative status. An interesting alternative for these studies is a detection of histone mRNA. As the synthesis of histones is tightly coupled with DNA replication during S-phase of the cell cycle, histone mRNA level is a specific marker of S-phase cells. Furthermore, a short-lived transcript guarantees accurate estimation of S-phase cell pool at the moment of tissue fixation. The progress in molecular biology techniques during the last decade made possible the use of in situ hybridization, especially its non-radioactive version, in routine laboratory services. This technique can be successfully applied to detection of histone mRNA in routinely processed tissues. Advantages and limitations of such approach in tumour proliferation studies are discussed.

Brain Neoplasms↗
PubMed1989

Effects of thyroparathyroidectomy and of thyroxin and calcitonin on the tissue distribution of twelve elements in the rat.

The effects of thyroparathyroidectomy (TPTY) and of replacement therapy using thyroxin (T4) and calcitonin (CT) on the tissue distribution of elements were studied in the rat under semichronic conditions. The elements Na, K, Ca, Mg, Fe, S, P, Rb, Sr, Mn, Cu, and Zn were determined in whole blood, plasma, brain, liver, heart, kidney, skeletal muscle, and bone. TPTY modified concentrations of all elements tested but only small changes were observed for K, Mg, S, and P. The mineral bone composition was slightly modified, 28 d after TPTY, whereas plasma was the most altered. The consequences of TPTY were corrected fairly well by T4 for Na, Cu, Zn, Fe, and S, and by CT for K, P, Rb but with less efficiency for Ca. This study revealed that hormones of the thyroid gland, mainly T4, play an important role in the plasma and tissue balance of elements. It is suggested that T4 participates in tissue fixation of Cu, Zn, and Fe and that CT influences phosphoremia and cellular Ca binding.

Animals↗
PubMed1998

Interference screw fixation of hamstring vs patellar tendon grafts for anterior cruciate ligament reconstruction.

The present study was designed to investigate the fixation strength of a quadruple semitendinosus-gracilis graft compared with a middle-third bone-patellar tendon-bone graft using a new interference screw developed to fix hamstrings grafts for ACL reconstructions (RCL Smith & Nephew Donjoy). Five pairs of human cadaveric knees from donors with a mean age of 43 (range 33-52) years were used. One knee of each pair was randomly allocated to be reconstructed on the femoral side with a semitendinosus-gracilis graft from the same donor using RCL screw fixation. As the control, the contralateral knee was correspondingly reconstructed with a bone-patellar tendon-bone graft using the same interference screw. The grafts were pulled out at a velocity of 30 mm/s by an axially applied load using a MTS machine. The mean (SD) failure load for the bone-patellar tendon-bone graft fixations was 505 (25) N, 110% stronger than the mean failure load for the semitendinosus-gracilis graft fixations, which was 240 (47) N (P = 0.003). The stiffness for the patellar tendon-bone graft fixations was 46 (11) N/mm, 120% stiffer than the semitendinosus-gracilis graft fixations, which was 22 (11) N/mm (P = 0.01). This study shows that the interference screw principle used for ACL reconstructions with hamstrings tendons is inferior to that for bone-patellar tendon-bone reconstructions although the screw was developed especially for soft-tissue fixation in bone tunnels.

Adult↗
PubMed1987

The influence of fixation on the morphology of mouse epidermis. A light and electron microscopical study with special reference to "dark cells" and epidermal carcinogenesis.

Different opinions exist on the normal ultrastructure of the epidermis including the significance of so-called basal dark cells. Thus, the dark cells are still assumed to be key elements in experimental skin carcinogenesis. We therefore explored the effects of tissue fixation on the ultrastructure of the epidermis. Untreated normal hairless mouse skin was processed for transmission electron microscopy with two different sets of fixatives, applied either by perfusion-immersion or immersion fixation only. The morphology of both the basal and the lower suprabasal layers of the epidermis, including the extracellular space, the shape and volume of the cells, their electron density, and the organisation of some of the organelles, were profoundly affected by the choice of fixatives. The non-keratinocytes showed comparable changes, including the appearance of a dark phenotype. The incidence of small electron-dense keratinocytes (dark cells) and the nature of their ultrastructure changed markedly with the fixation procedure. We were not able to identify undifferentiated dark cells. The pattern of changes and the quality of the morphological picture were almost unaffected by the mode of fixation. The upper suprabasal and the cornified layers appeared to be more or less unaltered by the change in fixatives and the method of application. The vehicle osmolality of the primary fixative was found to be mainly responsible for the ultrastructural appearances. A low vehicle osmolality may be responsible for the occurrence of the dark cell phenomenon, by inducing swelling artefacts of many cells with compression of some neighbouring cells.

Animals↗
PubMed1992

Quantitative immunohistologic study of lip biopsies. Evaluation of diagnostic and prognostic value in Sjögren's syndrome.

In a group of 45 patients with Sjögren's syndrome (SS) and 80 controls the high specificity (95%) and sensitivity (100%) of a recently proposed bivariate quantitative immunohistologic (QIH) criterion for SS, based on percentages of IgA and IgG-containing plasma cells in labial salivary gland (LSG) tissue, was confirmed. The best univariate QIH criterion for discrimination between LSG biopsies of SS patients and controls appeared to be based on the percentage of IgA containing plasma cells, and had a specificity of 99% and a sensitivity of 96%. A criterion based only on the percentages of IgM-containing plasma cells, proposed in another recent study, resulted in a high number (31%) of false negatives. Interobserver reproducibility of QIH diagnoses was excellent. Moreover it was demonstrated that accuracy, precision and the interobserver reproducibility of plasma cell counting depends on the choice of tissue fixation and immunohistologic staining procedure. The combination of formol sublimate fixation and peroxidase anti-peroxidase procedure appeared to be the best combination for QIH examination. Furthermore, in 2 SS patients systemic monoclonal IgM/kappa gammopathy was preceded by high predominance of IgM and kappa containing plasma cells in the plasma cellular infiltrate of the LSG tissue.

Biopsy↗
PubMed2006

Anterior cruciate ligament reconstruction: a new technique for Achilles tendon allograft preparation.

We describe a new technique in Achilles tendon allograft preparation for use in anterior cruciate ligament (ACL) reconstruction that allows for secure bony interference fixation on each side of the joint and aperture fixation for all patients. In addition, preparation of the graft in this manner avoids some problems that are frequently encountered with patellar tendon allografts, including graft tunnel mismatch and limited availability. Previous studies have reported successful results with Achilles tendon allograft use in ACL reconstruction with soft tissue fixation in the tibial tunnel. Bony interference fixation on the tibial side can be achieved by suturing a free bone plug to the tendon end of an Achilles allograft. We use a 9-mm circular oscillating saw to harvest a free 30-mm length bone plug from the remaining calcaneal bone block. This is then sutured directly to the tendon end of a bone-Achilles tendon allograft with the use of No. 1 nonabsorbable suture placed through 3 equally spaced drill holes in the free bone plug. Tendon length between the bone plugs can be individually set for each patient at a distance equivalent to the length of the native ACL (intra-articular distance between the femoral and tibial tunnels). After graft passage, the construct is tensioned and secured with interference screws, similar to a traditional bone-patellar tendon-bone graft. The senior author (S.G.) has performed 40 procedures with excellent results and reports no cases of tibial fixation failure. Biomechanical and long-term follow-up studies are in progress.

Achilles Tendon↗
PubMed1990

Herbert screw fixation of osteochondral fractures about the knee.

Osteochondral fracture of the patella or femoral condyle may occur with patella dislocation. Accepted treatments of these fractures are excision or replacement. In this paper eight cases are described in which the Herbert Bone Screw system was used for fixation. Normal knee function was regained within 6 months and there were no reported recurrent dislocations. The advantages of this technique are that it is simple, there is little cartilage damage done during insertion, the screw is entirely buried and therefore does not interfere with the soft tissues, fixation is rigid enough to allow for the application of continuous passive motion to aid in cartilage repair, and re-operation for removal is not necessary.

Adolescent↗
PubMed1992

Structure of rat kidneys following microwave accelerated fixation.

In contrast to fixation of tissue in externally heated fixative, microwave-irradiation can generate uniform internal heat, which is of utmost importance for successful fixation of biological tissue. To evaluate the effectiveness of microwave-accelerated chemical fixation, we compared the structure of rat kidney fixed by a conventional method and a microwave-accelerated method, by scanning and transmission electron microscopy. Following perfusion, rat kidney pieces 1-2 mm in size were irradiated in Karnovsky's fixative in a domestic Amana microwave oven, till the temperature of the fixative reached 45-50 degrees C. For conventional fixation, tissue pieces were fixed overnight at room temperature in the same fixative. Both types of samples were processed further for electron microscopy using identical protocols. The microwave fixed samples showed excellent preservation of structure comparable to the samples fixed by the conventional method. Glomeruli and the renal tubules showed normal morphology with no cellular swelling. The cytoplasm and nuclear matrix of the epithelial cells was uniformly dense. Other fixation sensitive organelles like mitochondria and Golgi apparatus showed superior preservation with continuous membranes. These results demonstrate that microwave accelerated chemical fixation results in excellent preservation of tissue structure, reduces processing time significantly and is therefore a practical alternative to conventional protocols.

Animals↗
PubMed1975

A postcoupling method for the demonstration of N-acetyl-beta-D-glucosaminidase in unfixed frozen tissue sections.

The localization and activity of the lysosomal enzyme, N-acetyl-beta-glucosaminidase, has been studied in unfixed frozen sections of tissues from normal and hemorrhaged rats with the use of a modified postcoupling technique. Discrete localization of the end product of the reaction was achieved in this method by the incorporation of 20% (w/v) polyvinyl alcohol (molecular weight 14,000) in the incubation medium. Advantages of the present method include the ability to overcome the inhibitory effects on enzyme activity of both tissue fixation and the presence of diazonium salts in the incubation medium. The staining reaction obtained with this technique demonstrates the enzyme activity at specific cytoplasmic sites within cells and has a wider applicability to the comparative study of N-acetyl-beta-glucosaminidase activity in normal and injured tissues.

Acetylglucosaminidase↗
PubMed1985

A method for silver impregnation of mammary myoepithelia.

Histochemical methods for microscopic visualization of mammary myoepithelial cells all yielded considerable variation in completeness of myoepithelial cell staining. Although extremely variable, silver impregnation occasionally gave tissue sections containing myoepithelia having excellent microanatomical detail and contrast with other tissue elements. Consequently, sources of variation in the silver technique were considered. Composition of the tissue fixative and pH of the silver impregnating solution were most critical. A final method is presented which gives consistent, complete silver impregnation of myoepithelia, where both the cell body and cell processes are clearly evident. The staining procedure is not light sensitive, nor is acid cleaning of glassware necessary. Tissue sections from lactating mouse, rat, hamster and goat are presented; tissue from other species should stain as well. The procedure should greatly facilitate the study of the function of myoepithelial cells and the visualization of these cells in mammary pathology.

Animals↗
PubMed1990

FITC-dextran as a probe for endosome function and localization in kidney.

Fluorescein isothiocyanate (FITC)-labeled endosomes were localized in kidney epithelial cells after tissue fixation and sectioning, and specific membrane transport properties of isolated endocytic vesicles were measured using the same probe. Rats were infused intravenously with 10 kDa FITC-dextran, and kidneys were fixed with paraformaldehyde lysine periodate. FITC-labeled vesicles were visualized in semithin (1 micron) frozen sections of excised tissue by epifluorescent microscopy and by electron microscopy after a photoconversion reaction. Most FITC-labeled endosomes were apically located in epithelial cells lining the urinary tubules. By immunocytochemistry the anti-lysosomal glycoprotein LGP 120 was absent from most of the FITC-labeled vesicles, although some colocalization was noted. The limiting membrane of FITC-labeled endosomes contained a vacuolar proton pump (pHmin = 6.23 +/- 0.033) and a water channel (osmotic water permeability coefficient, Pf = 0.052 +/- 0.005 cm/s) and was highly permeable to ethylene glycol and urea but relatively impermeable to glucose. Methods allowing the attribution of specific membrane functions to vesicles that can be visualized in the apical endocytic pathway of epithelial cells should be of general use for the study of endocytic pathways in a variety of systems.

Adenosine Triphosphate↗
PubMed2006

Expression of the multidrug transporter MRP2 in the blood-brain barrier after pilocarpine-induced seizures in rats.

Multidrug resistance proteins (MRPs; symbol ABCC) are membrane glycoproteins that mediate the ATP-dependent export of a wide range of substrates from cells and thereby affect the bioavailability and disposition of many drugs. MRP2 (ABCC2) is expressed on the apical domain of hepatocytes, enterocytes of the proximal small intestine, and proximal renal tubular cells, but its location in the brain is a matter of debate. Most previous studies failed to determine MRP2 mRNA or protein in the brain or cell preparations from the brain of different species including humans. Based on our previous experience with the drug efflux transporter P-glycoprotein, we evaluated whether the immunohistochemical determination of MRP2 expression is sensitive to fixation and staining variables. Furthermore, we examined whether the MRP2 protein is overexpressed after experimentally induced seizures in rats, using the pilocarpine model of temporal lobe epilepsy. The MRP2 expression in the liver was used as positive control. MRP2 deficient TR- rats were used as negative controls. Despite various modifications in tissue fixation and immunohistochemical staining as well as use of different commercially available MRP2 antibodies, we never observed any unequivocal MRP2 staining in the brain of normal rats. However, after a pilocarpine-induced convulsive status epilepticus, clear MRP2 staining became visible in brain capillary endothelial cells and, less frequently, perivascular astroglia and neurons in various brain regions. In view of our recent data on brain access of antiepileptic drugs in MRP2 deficient TR- rats, seizure-induced over-expression of MRP2 in the blood-brain barrier is likely to impair drug penetration into the brain, thereby contributing to drug resistance in epilepsy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Immunohistochemical markers for the rodent immune system.

The responses to insults including chemical toxins, irradiation and infectious agents involve morphologic, biochemical and molecular changes in the immune system. The changes in specific tissues and cells often can be detected by histopathology and its associated field of immunohistochemistry (IHC). Cells normally express specific proteins (antigens) that can be detected by IHC. When responses to xenobiotics occur, cells often up or down regulate proteins. The art of IHC requires specialized procedures for detection of antigens. Fixation, tissue processing, immunoreactions and antigen retrieval methods are important elements of IHC. We review the antibodies, their sources, use of frozen or fixed paraffin-embedded tissues and specific IHC methods including antigen retrieval and illustrate how they can be effectively used to characterize the immunotoxicologic effects of agents.

Animals↗
PubMed1987

Ultrastructural and immunofluorescent detection of herpes simplex virus after embalming and burial.

The practice of embalming preserves body tissues, and embalmed bodies may resist decay processes for many decades with relatively little change. As the chemicals used for embalming are poisonous to microorganisms, bacterial and viral cultures are futile after such funerary procedures are performed. However, embalming may act as a virtual tissue fixative, especially with arterial perfusion, and identification methods other than culture may be used to detect and identify pathogenic organisms. In the case presented here, a death from a fulminating, but unidentified, illness in a young girl was successfully diagnosed as herpes hepatitis by immunofluorescent and electron-microscopic studies of tissue obtained 3 weeks after she was embalmed and interred. Routine embalming and burial should not eliminate these diagnostic procedures from consideration in specific situations where potentially useful information may be realized.

Autopsy↗
PubMed2002

Tissue microarrays are an effective quality assurance tool for diagnostic immunohistochemistry.

There has been considerable variability in the reported results of immunohistochemical staining for some diagnostically relevant antigens. Our objectives in this study were to (1) use a multitumor tissue microarray with tissue from 351 cases received in our department, representing 16 normal tissues and 47 different tumor types, to compare immunohistochemical staining results in our laboratory with published data, using a panel of 22 antibodies; (2) assess interlaboratory variability of immunohistochemical staining for S-100 using this microarray; and (3) test the ability of hierarchical clustering analysis to group tumors by primary site, based on their immunostaining profile. Tissue microarrays consisting of duplicate 0.6-mm cores from blocks identified in the hospital archives were constructed and stained according to our usual protocols. Antibodies directed against the following antigens were used: B72.3, bcl-2, carcinoembryonic antigen, c-kit, pankeratin, CD 68, CD 99, CK 5/6, CK 7, CK 8/18, CK19, CK 20, CK 22, epithelial membrane antigen, estrogen receptor, melan-A, p53, placental alkaline phosphatase, S-100, synaptophysin, thyroid transcription factor-1, and vimentin. Staining results on the array cases were compared with published results, and hierarchical clustering analysis was performed based on the immunohistochemical staining results. Unstained slides of the multitumor tissue microarray were sent to five other diagnostic immunohistochemistry laboratories and stained for S-100 protein. The staining results from the different laboratories were compared. Staining results using our current methods and samples from our laboratory were compatible with those described in the literature for most antigens. Placental alkaline phosphatase staining was not specific with our protocol, showing staining of a broad spectrum of different tumors; this finding initiated a review of our recent requests for placental alkaline phosphatase immunostaining and revealed two instances in which placental alkaline phosphatase positivity was incorrectly interpreted as evidence of a germ cell tumor. S-100 staining was less sensitive but more specific for the diagnosis of melanoma or neural tumor in our laboratory, compared to some published reports. Assessment of interlaboratory variability of S-100 immunostaining showed that there was more frequent staining of carcinomas in some laboratories, resulting in decreased specificity of S-100 staining in distinguishing melanoma from carcinoma. Hierarchical clustering analysis showed a strong trend for tumors to cluster by tissue of origin, but there were significant exceptions. We conclude that multiple-tumor microarrays are an efficient method for assessing the sensitivity and specificity of staining with any antibody used diagnostically. As a tool for quality assurance, they offer the advantage of taking into account local differences in tissue fixation, processing, and staining. They also allow cost-effective assessment of interlaboratory variability in immunohistochemical staining. Results of hierarchical clustering analysis show the potential for panels of immunohistochemical stains to identify the primary site of metastatic carcinomas but also confirm the limitations of currently available antibodies in giving unequivocal tissue-specific staining patterns.

Biomarkers, Tumor↗