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PubMedDate not supplied

Comparison of in vivo and in vitro demineralized dentin with phosphoric and maleic acid.

This article evaluated possible differences between dentin conditioned in vivo and in vitro with 10 percent maleic acid or with 36 percent phosphoric acid. Semispherical Class V cavities were prepared in vivo and in vitro at the cementum-enamel junction and were divided into four groups. After etching procedures, the in vivo samples were extracted and fixated in 10 percent buffered formaldehyde. Both the in vivo and in vitro samples were then fractured in two parts along their long axis, critical-point dried, and subsequently examined with SEM. Both the acids removed completely the smear layer and demineralized the dentin, leaving a layer of collagenous network. No morphological differences were found between in vivo and in vitro dentin samples.

Acid Etching, Dental↗
PubMedDate not supplied

Immunoreactivity to MIB-1 in breast cancer: methodological assessment and comparison with other proliferation indices.

The recent availability of the monoclonal antibody MIB-1 (which is able to detect the human nuclear cell proliferation-associated antigen Ki-67 even on formalin-fixed, paraffin-embedded sections, microwave-treated and routinely processed for immunohistochemistry) could open new avenues for validation of the clinical role of tumour cell proliferation on large, consecutive and unselected series of human tumours. However, the routine use of such a marker requires a methodological standardization as well as the comparative assessment of some technical and biological aspects. The MIB-1 index was determined in parallel samples from 50 consecutive invasive breast carcinomas processed with different fixatives for different times. The median values of MIB-1 indices following 2, 6 and 24 h of formalin fixation were similar (29.4%, 30.6% and 29.7%, respectively) and consistent with those reported in the literature; squared linear regression coefficients were 0.99. The median values of MIB-1 indices were markedly lower in Bouin-fixed, paraffin embedded, and in frozen samples (20.0% and 19.8%, respectively), with a poor correlation coefficient with the values detected following formalin fixation (R2 = 0.456). Moderate and poor correlations were observed between Ki-67 index and MIB-1 detected on frozen (rs, 0.78) or formalin-fixed, paraffin-embedded samples (rs, 0.47) and a minimal concordance was observed between TLI and MIB-1 or Ki-67 (rs, 0.25 and 0.22, respectively). Our results indicate interference of the fixative type on immunoreactivity to MIB-1 and also suggest that Ki-67 and MIB-1 reacted with different epitopes of the same antigen.

Antibodies, Monoclonal↗
PubMed1994

Anisotropic elasticity and strength of glutaraldehyde fixed bovine pericardium for use in pericardial bioprosthetic valves.

Uniaxial tensile tests were performed on glutaraldehyde fixed bovine pericardial strips prepared from chemically modified pericardial samples. These samples originated from an area which demonstrated anisotropic mechanical properties in the native material and which is suitable for the construction of leaflets for pericardial bioprostheses. After glutaraldehyde fixation the tissue had retained its anisotropicity in stiffness and strength in two orthogonal directions. In the range of the functional stresses for a heart valve leaflet (< 1 MPa) the unconstrained fixation regime had modified the initial anisotropic elastic behavior into a more isotropic one. The implications of these findings are that leaflets manufactured from bovine pericardium can be made to resemble, to a degree, the well known anisotropy found in two orthogonal directions in natural human heart valve leaflets, or porcine bioprosthetic heart valve leaflets.

Animals↗
PubMed1986

Determination of lipid loss during aqueous and phase partition fixation using formalin and glutaraldehyde.

Phase partition fixation permits fixation of tissue in a nonaqueous environment, thus eliminating osmotic effects. It was shown in an earlier investigation that retention of protein in liver blocks can be improved by phase partition fixation. By using radioisotopic labeling techniques, the effects of phase partition fixation on lipid retention during fixation, dehydration, and clearing have been determined and compared with those of standard aqueous fixation techniques. In this article we show that retention of total lipid in liver blocks following phase partition fixation using formalin was comparable to or better than that with aqueous formalin fixation and processing. Fixation with glutaraldehyde using phase partition fixation resulted in somewhat greater loss of total lipid than that observed for aqueous buffered glutaraldehyde-fixed blocks.

Acetates↗
PubMed2004

Ultra-rapid microwave-stimulated tissue processing with a modified protocol incorporating microwave fixation.

AIMS: To develop an ultra-rapid microwave (MW)-stimulated histoprocessing protocol that incorporates MW fixation and produces consistent, high quality sections. METHODS: A range of fresh autopsy tissues was divided into three groups composed of equal numbers of small and large tissue blocks. Group 1 tissues were fixed for 8 hours in 4% buffered formaldehyde and processed in a conventional tissue processor through a 15-hour cycle. Group 2 tissues were processed in a MW histoprocessor according to the manufacturer's recommended protocol of irradiation in a proprietary reagent before embedding in paraffin. Group 3 tissues were processed with our protocol that incorporated the addition of MW fixation in 4% buffered formaldehyde and MW irradiation in isopropyl alcohol after irradiation in the proprietary reagent. RESULTS: The manufacturer's protocol resulted in 'grey' and 'wet look' artefacts in large tissue blocks. These artefacts did not occur in our protocol which produced sections that were indistinguishable from those obtained with conventional 23-hour processing. Histochemical and immunohistochemical stains were also indistinguishable and the tissue blocks cut very smoothly. CONCLUSIONS: The incorporation of an additional step to ensure adequate tissue fixation and one to ensure optimal dehydration and clearing resulted in a MW histoprocessing protocol that produced consistent results for both large and small tissue blocks. Paraffin-impregnated blocks can be produced very rapidly from fresh small and large tissue blocks in 45 and 100 minutes, respectively.

Artifacts↗
PubMed1985

A recommended procedure for ultrastructural immunohistochemistry on small human tissue samples.

Various fixation and staining procedures for the demonstration of surface and cytoplasmic antigens have been described. An immunostaining procedure was sought that would allow the demonstration of these antigens, especially in small human tissue samples at the ultrastructural level. A modification and adaptation of the technique of Eldred, Zucker, Karten, and Yazula (J Histochem Cytochem 31:285, 1983) was applied on several varieties of human tissue, including liver, skin, and lymphoid tissue, using monoclonal and polyclonal antibodies in an indirect peroxidase procedure. In this way a reliable and generally applicable procedure was developed that satisfied the following demands: Use of a universal fixative that allows preservation of the antigenicity of various antigens; Adequate penetration of the tissue by the immunological reagents; Optimal preservation of subcellular structures; and Possibility to store the tissue samples for considerable periods of time.

Antibodies, Monoclonal↗
PubMed1993

Improved preservation of rat epididymal sperm for high-resolution low-voltage scanning electron microscopy (HR-LVSEM).

Various fixation protocols were used in an attempt to improve preservation of rat epididymal sperm for high-resolution low-voltage scanning electron microscopy (HR-LVSEM). Wash solutions and fixatives of different composition and osmolarity were tested. Paraformaldehyde and glutaraldehyde concentrations were varied between 0.5% and 3%. Ruthenium red was tested as an additive in both primary fixation and postfixation, or in postfixation alone. HR-LVSEM revealed various degrees of ruffing, folding, blebbing, and peeling off of the plasma membrane, as well as holes of different sizes. The plasma membrane overlying the acrosome and the connecting piece proved to be particularly sensitive to varying fixation conditions. Consistent topographical differences were revealed among the different domains over the sperm head. Most of the differences were considered to be artifacts. Their consistency, however, suggests that structural and biochemical differences exist either within the membrane or in the structures subjacent to the membrane. Primary fixation turned out to be less critical than postfixation. Preservation of a smooth plasma membrane without holes could only be achieved when primary fixation in low aldehyde concentrations, with or without ruthenium red, was followed by postfixation with OSO4 and 1,000 ppm ruthenium red. Examination of thin sections of the same material confirmed that even a considerable number of small holes are difficult to detect in transmission electron microscopy. These results show that with the recent increase in resolution of LVSEM there is need for further effort to improve sample processing.

Animals↗
PubMed1975

[The unilateral emigration of leukocytes from conjunctival vessels (author's transl)].

The phenomenon of unilateral leukodiapedesis is demonstrated in the irritated conjunctiva of rabbits and rhesus-monkeys. The leukocytes will penetrate the vessel walls first in the neighbourhood of the irritation. This can be demonstrated during the first minutes after the onset of an inflammatory reaction only, i.e. prior to a general leukocytosis of the tissue. By fixation with glutaraldehyde or osmium-tetroxyde the dynamic movement can be visualized and the directions of the irritation and of the migration are obvious in the conjunctiva. So this tissue provides an excellent model to demonstrate the unilateral sticking and the directed migration of leukocytes through vessel walls, a fact to which references seem to be missing in the ophthalmopathologic and pathologic literature.

Animals↗
PubMed2006

Fixation methods can produce misleading artifacts in sperm cell ultrastructure of diploid and tetraploid Pacific oysters, Crassostrea gigas.

Spermatozoa from diploid and tetraploid Pacific oysters (Crassostrea gigas) were examined after anisotonic fixation. Morphological anomalies, such as membrane rupture, detached tails, and the formation of tail vesicles (typically associated with damage attributable to procedures such as cryopreservation) were observed; the Mantel-Haenszel Chi-square test indicated a strong association between the anomalies and fixative osmolality (P<0.001). The present study also indicated that media in a range of 800 to 1,086 mOsm/kg could be assumed to be functionally isotonic to Pacific oysters, and osmolalities below or above this caused severe cell damage. For example, the maximum volume of flagella obtained after hypotonic fixation was approximately twice the volume of the flagella in isotonic fixation. Sperm cell flagellar volumes after hypertonic fixation (1,110 mOsm/kg) were 32% smaller than those in isotonic fixation, and sperm heads were 25% smaller. Although the damage associated with anisotonic fixation was evident in all parts of the sperm cells, the most vulnerable locations were the plasma membrane and flagellum motor apparatus. The formation of tail vesicles after hypotonic fixation was also examined. Because of water uptake, oyster sperm became swollen in hypotonic fixative, and bending or coiling of the axoneme within the tail vesicles led to the appearance of multiple axonemal structures in cross sections when observed by transmission electron microscopy. This phenomenon might be generally misinterpreted as the presence of double tails. This and other fixation artifacts can lead to the misinterpretation of damage caused by cryopreservation in ultrastructure studies of sperm of aquatic species, especially those in marine species.

Animals↗
PubMed2006

Validation of the quality of histological images obtained of fresh and formalin-fixed specimens of esophageal and gastric mucosa by laser-scanning confocal microscopy.

BACKGROUND AND STUDY AIMS: We have previously reported the success of a method of virtual histology using laser-scanning confocal microscopy (LCM) in vitro on untreated fresh specimens obtained from the gastrointestinal mucosa. In the present study, we aimed to apply LCM to both fresh and formalin-fixed specimens, without additional treatment, in order to validate and compare the quality of the images obtained. METHODS: We obtained 18 specimens from 11 patients, either by endoscopic biopsy or following surgical resection. First, we observed the fresh, saline-immersed specimen with LCM using the Fluroview microscope (Olympus Co. Ltd., Tokyo, Japan). We then fixed the specimen with formalin and obtained further LCM images 1 hour, 3 hours, and 24 hours after fixation. Three independent observers observed the images and were asked to assess the origin of the samples, the treatment of the samples, the time after formalin fixation, and whether they showed benign or malignant lesions. We used kappa statistics to compare the agreement among the three observers in each of these four areas of interest. RESULTS: Between January and March 2003, we obtained 191 LCM images from 18 specimens. Thirty images were randomly selected for observation. The overall accuracy for differentiating between esophagus and stomach specimens was 96.6 %. The accuracy of differentiating normal from cancerous lesions was 92.2 %. The differentiation between saline-immersed and formalin-fixed specimens was 59.7 % accurate and the assessment of the time interval after formalin fixation was only 37.3 % accurate. The kappa statistics showed that there was strong interobserver agreement on the differentiation of specimen origin and of cancerous from benign lesions. However, there was no agreement among the observers on the method of specimen preparation or on the estimated time interval after formalin fixation. CONCLUSIONS: We concluded that images obtained from fresh specimens using LCM were of a quality good enough to make an accurate diagnosis of upper gastrointestinal carcinoma.

Biopsy↗
PubMed2000

Review article fine structure of the exocrine cells of rat sublingual gland revealed by rapid freezing and freeze substitution method.

The ultrastructure of mucous cells of rat sublingual gland processed by rapid freezing, followed by freeze substitution, was compared with that obtained by the standard chemical fixation technique. The rapid freezing method gave a very good preservation of membrane structure with round and discrete mucous droplets (granules) not showing any sign of coalescence. The cisterns of the Golgi apparatus and the trans Golgi network also were well preserved. Upon secretory stimulation by pilocarpine, mucous droplets were discharged by the usual mechanism of exocytosis. From all these findings it emerged that mucous cells had the same structural characteristics as serous cells. In the endpieces of rat sublingual gland prepared by the rapid freezing method, serous cells aligned with mucous cells around the central lumen, and no cap-like arrangement of serous cells (demilunes) was observed. Furthermore, computer reconstruction of stereo images from serial section light micrographs prepared by the rapid freezing method showed that, within a given endpiece, all serous cells had direct access to the lumen and that they were disseminated throughout it and not only in its fundus. From our observations it seems very likely that, at least in rat sublingual gland, serous demilunes are an artificial product caused by the compression exerted on serous cells by the mucous cells distended during the conventional fixation procedure.

Animals↗
PubMed1997

Comparative evaluation of unfixed and fixed human neutrophils for determination of antineutrophil cytoplasmic antibodies by indirect immunofluorescence.

BACKGROUND: Antineutrophil cytoplasmic antibodies (ANCAs) are found in the sera of patients with vasculitides and ulcerative colitis. Using indirect immunofluorescence on ethanol fixed neutrophils, ANCAs can be divided into two types: those that give a cytoplasmic staining pattern (C-ANCA) and those that give a perinuclear staining pattern (P-ANCA). Some studies have indicated that the perinuclear staining pattern might be an artefact of alcohol fixation. AIMS: To observe any changes seen in the ANCA staining pattern using indirect immunofluorescence on unfixed neutrophils or neutrophils that had been fixed by ethanol, acetone, or paraformaldehyde. In addition, the effects of the different fixation methods on the sensitivity of the indirect immunofluorescence test were evaluated. METHODS: Twenty one sera from patients with ulcerative colitis and 19 from healthy controls were studied. In addition, 17 sera from patients with vasculitides, including eight with proteinase 3 (PR 3) positive C-ANCA and nine with myeloperoxidase (MPO) positive P-ANCA were included in the study. Antineutrophil cytoplasmic antibodies were analysed by indirect immunofluorescence on unfixed neutrophils or cells fixed by ethanol, acetone, or paraformaldehyde. RESULTS: All the ulcerative colitis associated ANCA positive sera presented a peri-nuclear staining pattern on both unfixed and fixed cells. The acetone and paraformaldehyde fixations decreased the ulcerative colitis P-ANCA titres. Paraformaldehyde fixation also decreased the MPO positive P-ANCA titres. In indirect immunofluorescence, the staining patterns of all the eight PR 3 positive C-ANCA sera and eight of the nine MPO positive P-ANCA sera did not change, even if unfixed or fixed cells were used. CONCLUSIONS: The ANCA staining patterns are not affected by the fixation method used, and are the same as when unfixed neutrophils are used; this suggests that the P-ANCA pattern is not an artefact of alcohol fixation. Furthermore, this study confirms that on ethanol fixed neutrophils, the antigen of ulcerative colitis associated P-ANCA is better exposed than on acetone or paraformaldehyde fixed cells.

Acetone↗
PubMed2001

Detection of the Mr 110,000 lung resistance-related protein LRP/MVP with monoclonal antibodies.

The Mr 110,000 lung resistance-related protein (LRP), also termed the major vault protein (MVP), constitutes >70% of subcellular ribonucleoprotein particles called vaults. Overexpression of LRP/MVP and vaults has been linked directly to MDR in cancer cells. Clinically, LRP/MVP expression can be of value to predict response to chemotherapy and prognosis. Monoclonal antibodies (MAbs) against LRP/MVP have played a critical role in determining the relevance of this protein in clinical drug resistance. We compared the applicability of the previously described MAbs LRP-56, LMR-5, LRP, 1027, 1032, and newly isolated MAbs MVP-9, MVP-16, MVP-18, and MVP-37 for the immunodetection of LRP/MVP by immunoblotting analysis and by immunocyto- and histochemistry. The availability of a broader panel of reagents for the specific and sensitive immunodetection of LRP/MVP should greatly facilitate biological and clinical studies of vault-related MDR.

Animals↗
PubMed1993

Pathological specimens of rat temporal bone--comparison between different fixative solution.

We investigated the difference in temporal bone specimens fixated by three sorts of fixative solution (10% formalin fixative, Wittmaack's fixative and Heidenhein-SuSa fixative). 1. 10% Formalin's fixative solution We found many pinkish precipitates, which are stained by hematoxy-eosin, in the scala media of the cochlea. Same substance is found in the perilymphatic space and endolymphatic space in the vestibulum. The fusion of the cells is indicated in the tectorial membrane, inner sulucus cell, outer sulucus cell especially in the apical turn of the cochlea. 2. Wittmaack's fixative solution The detachment of inner and outer sulucus cells are found from basilar membrane at the basal turn of the cochlea. The tendency in convex and concave form of the Reissner's membrane is most remarkable in these three fixative solution. 3. Heidenhein-SuSa fixative solution In this solution, no precipitates, no fusion of cells are found. No detachment of the inner or outer sulucus cells is also found. This study indicates that Heidenhein-SuSa fixation is most excellent fixative method.

Animals↗
PubMed2002

Hypothesis for the influence of fixatives on the chromatin patterns of interphase nuclei, based on shrinkage and retraction of nuclear and perinuclear structures.

Nuclear chromatin patterns are used to distinguish normal and abnormal cells in histopathology and cytopathology. However, many chromatin pattern features are affected by aspects of tissue processing, especially fixation. Major effects of aldehyde and/or ethanol fixation on nuclei in the living state include shrinkage, chromatin aggregation and production of a 'chromatinic rim'. The mechanisms of these effects are poorly understood. In the past, possible mechanisms of fixation-induced morphological change have been considered only in terms of the theoretical model of the nucleus, which involves only a random tangle of partly unfolded chromosomes contained within the nuclear membrane. Such a model provides no basis for chromatin to be associated with the nuclear envelope, and hence no obvious clue to a mechanism for the formation of the 'chromatinic rim' in fixed nuclei. In recent years, two new models of nuclear structure have been described. The nuclear membrane-bound, chromosomal-domain model is based on the discoveries of chromatin-nuclear membrane attachments and of the localisation of the chromatin of each chromosome within discrete, exclusive parts of the nucleus (the 'domain' of each partly unfolded chromosome). The nuclear matrix/scaffold model is based on the discovery of relatively insoluble proteins in nuclei, which it suggests forms a 'matrix' and modulates gene expression by affecting transcription of DNA. Here, a hypothesis for fixation-associated chromatin pattern formation based mainly on the first model but partially relying on the second, is presented. The hypothesis offers explanations of the variations of appearance of nuclei according to fixation (especially air-drying versus wet-fixation with formaldehyde, glutaraldehyde or ethanol); the appearances of the nuclei of more metabolically active versus less metabolically active cells of the same type; the appearances of nuclei after fixation with osmium tetroxide; and of the marked central clearing ('egg-shell' or 'orphan Annie' appearance) of tumour nuclei of papillary carcinoma of the thyroid gland. A similar process may underlie the phenomenon of 'chromatin margination' seen in apoptosis. Various tests of the hypothesis, such as time-lapse confocal microscopy of living nuclei during fixation, are suggested. The significance of the theory is that it suggests that chromatin patterns could be investigated in terms of qualitative and quantitative aspects of nuclear components, and hence be related to the results of studies of the structure and function of nuclei in health and disease.

Cell Nucleus↗