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PubMed1985

Comparative usefulness of tissue fixatives for in situ viral nucleic acid hybridization.

Traditionally tissues for in situ hybridization of viral nucleic acid have been small pieces obtained from laboratory rodents, and fixatives that are designed for electron microscopy, such as periodate-lysine-paraformaldehyde (PLP) can handle them adequately. However, these fixatives have limited penetrating ability and may produce no appreciable hardening, so alternative fixation methods were evaluated. The intention was to determine whether fixatives adequate for bulky tissues such as whole or halved pig and cow brains would also be compatible with in situ hybridization. Various fixatives were evaluated using a system of intracranial inoculation of BALB/c mice with pseudorabies virus (PRV) followed by in situ hybridization of brain tissue sections with a 35S-labeled PRV DNA probe. Loss of tissue sections was a major problem, particularly with PLP and formalin, but positive results were obtained with five fixatives tested. Cellular morphology was especially good with PLP and with a modification of Carnoy's fluid, MOCA fixative. An incidental but important observation was that formalin is compatible with in situ hybridization. Retroactive studies of viral diseases using routinely processed blocks of tissue (formalin-fixed, paraffin-embedded) are therefore conceivable.

Acetates↗
PubMedDate not supplied

Soft tissue fixation to bone. A biomechanical analysis of spiked washers.

The initial fixation strengths of two spiked-washer designs were evaluated using human femurs and fascia lata tissue. Fascia lata was attached to the femur using the fixation devices, and then each femur-washer-fascia lata complex was loaded in tension to failure. Load-elongation curves were recorded, the initial failure load, initial failure displacement, stiffness, ultimate load, and ultimate displacement were determined for each test, and failure modes were recorded. Results indicated that the 6-spike design provided superior initial fixation strength in the 19-mm diameter size. This washer design was then compared with two commercially available fixation devices: the spiked AO washer and soft tissue fixation plate. Fixation provided by the prototypal washer design was not different in most instances from that provided by the AO fixation devices. Based on these results, important design characteristics for soft tissue spiked washers are identified and discussed.

Acetals↗
PubMed2005

The effect of tissue fixation and processing on breast cancer size.

Precise measurement of an invasive breast cancer is crucial for pathological staging and subsequent patient management. Formalin fixation and histological processing may change tissue size, but there is no agreement on which state of the specimen, fresh or fixed, should be used for final tumor measurement. To determine the influence of fixation and processing on breast tumor size, a specific 1-dimensional measurement from 50 invasive breast tumors was recorded in fresh, fixed, and processed/mounted states. Tumors varied in maximum measured dimension from 4 to 20 mm and contained 10% to 90% estimated fibrous tissue (mean, 52.8%). In 96% of cases, there was no difference in measured size between fresh and fixed states. After final processing and mounting, a decrease in size from initial fresh measurement was noted in 40% of cases (mean difference, 2.4 mm; maximum difference, 7 mm). In 9 cases (18%), the measured size increased by a maximum of 3 mm (mean, 1.7 mm) after processing/mounting. Twenty-one cases (42%) showed no change in measurement during the entire fixation and processing protocol. Increases in measured size were attributed largely to tissue expansion during histological sectioning/mounting. One can arguably measure the size of an invasive breast cancer from either the fresh or fixed state without affecting accuracy, but caution should be exercised in relying solely on the microscopic measurements.

Adenocarcinoma↗
PubMed1985

Limitations of postmortem assessment of human coronary artery size and luminal narrowing: differential effects of tissue fixation and processing on vessels with different degrees of atherosclerosis.

Numerous studies have utilized histologic sections of coronary arteries as the standard for testing the validity of the angiographic determination of coronary artery dimensions. However, little attention has been given to artifactual dimensional changes that occur during fixation and histologic processing of tissues (dehydration, clearing, embedding, sectioning and staining). Using planimetric techniques, the dimensional changes that occurred with fixation and processing were quantitated in 61 coronary artery segments with minimal or moderate to severe atherosclerosis obtained from 12 patients studied at autopsy. In vessels with minimal atherosclerotic narrowing, fixation and processing resulted in a decrease in total vessel cross-sectional area and luminal cross-sectional area (p less than or equal to 0.05), whereas absolute wall area (total vessel cross-sectional area minus luminal cross-sectional area) did not change (p = NS). These disproportionate changes resulted in an alteration in the relation between lumen and wall areas so that luminal cross-sectional area decreased from 47.6 +/- 8.5% of the total vessel cross-sectional area observed before fixation to 36.2 +/- 7% after processing (p less than or equal to 0.05). In vessels with moderate to severe atherosclerosis, both the total cross-sectional area and wall area decreased after fixation and processing (p less than or equal to 0.05), but luminal area did not change (p = NS). As a result, the percent luminal cross-sectional area in these vessels increased from 21.1 +/- 10.1% before fixation to 28.7 +/- 9.7% after processing (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Arteriosclerosis↗
PubMed1981

Tissue fixation and staining by osmium tetraoxide: a possible role for alkaloids.

In vitro studies on the reactions of osmium tetraoxide, OsO4, with isoquinoline, pyridine, quinuclidine, and a series of structurally related alkaloids suggest that these species, under the conditions of normal tissue staining and fixation, are potentially osmiophilic. The structure of the products and their reactions with unsaturated substrates are described. The relevance of these results to the process of tissue staining and fixation of plant tissues containing alkaloids by OsO4 is discussed.

Alkaloids↗
PubMed2002

Effect of tissue fixatives on the immunohistochemical expression of ABH blood group isoantigens.

Immunohistochemical analysis of ABH blood group isoantigens has been shown to be a useful ancillary technique for resolving problems associated with specimen mix-ups in the daily practice of surgical pathology. However, the effects of different fixatives on the expression of these antigens in paraffin-embedded tissues are not known. Therefore, the effects of seven different fixatives on the immunohistochemical expression of ABH blood group isoantigens were studied in tissues from several organs. The following fixatives were used: acetone, 70% ethanol, B5, Bouin, Carnoy, methanol, and 10% formalin. After fixation for 6, 12, and 72 hours, the tissue blocks were embedded in paraffin, and immunohistochemistry was performed on 4 microm-thick tissue sections using monoclonal antibodies to blood group isoantigens (A, B, and H) and the avidin-biotin detection method. Also, immunostaining was performed on step tissue sections with and without antigen retrieval using citrate buffer at pH 6.0. The expression of the blood group isoantigens was concordant with the blood group of the patient in all the cases studied, irrespective of the fixative and time of fixation. However, in the absence of antigen retrieval, the intensity of the staining reaction was diminished. These results showed that irrespective of the fixative used, immunohistochemical staining of paraffin-embedded tissue sections with ABH blood group antibodies is a rapid, reliable, and cost-effective method for sorting out interpretative problems of tissue contaminants (floaters) and specimen mix-ups in surgical pathology.

ABO Blood-Group System↗
PubMed2002

Improved detection of simian immunodeficiency virus RNA by in situ hybridization in fixed tissue sections: combined effects of temperatures for tissue fixation and probe hybridization.

In situ hybridization detection of viral RNAs in formaldehyde-fixed tissue specimens is used frequently to characterize the extent of viral replication within host tissues. The ability to determine the level of expression of viral RNAs in situ is dependent upon many factors including the extent of cross-linking during fixation, the pretreatment regimen utilized to relieve the effects of cross-linking, and the hybridization and wash protocols. In efforts to improve our ability to detect cells infected productively by simian immunodeficiency virus (SIV) in rhesus macaque tissues, the effects of unconventionally high (40 degrees C) and more standard low (4 degrees C) temperature fixation in 4% paraformaldehyde/phosphate buffered saline were tested empirically on in situ hybridization signals. In addition, hybridization temperatures ranging between 37 and 75 degrees C were utilized to determine the optimal hybridization conditions for detection of SIV productively infected cells. Fixation conditions of 40 degrees C and hybridization conditions of 50-55 degrees C were identified as providing the greatest sensitivity for detecting RNA(+) cells and for quantitating the signal per cell, while still allowing antigenic epitopes to be detected by immunohistochemical staining. These data indicate that the signal intensity following in situ hybridization for viral RNAs is dependent upon the combined effects of tissue fixation and in situ hybridization temperatures.

Animals↗
PubMed2002

Evaluation of non-formalin tissue fixation for molecular profiling studies.

Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analyzed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. Complete protocols and additional discussion of relevant issues are available on an accompanying website (http://cgap-mf.nih.gov/).

Actins↗
PubMed1997

Sporicidal activity of chemical and physical tissue fixation methods.

AIMS: The effects of alcohol based fixation and microwave stimulated alcohol fixation were investigated on spores of Bacillus stearothermophilus and Bacillus subtilis (var. niger). METHODS: Spores were exposed to 10% formalin, or different concentrations of various alcohol containing fixatives (Kryofix/Spuitfix). Adequate controls were also set up in conjunction with the test solutions. The spores were immersed with and without adjunctive microwave stimulation in the various solutions tested. Possible surviving spores were recovered in revival broth and after incubation, and Gram staining viable counts were performed. RESULTS: Alcohol based fixatives did not have a sporicidal effect on B stearothermophilus or B subtilis (var. niger) spores, and microwave stimulated alcohol fixation at 450 W and up to 75 degrees C did not have a sporicidal effect. CONCLUSIONS: When alcohol based fixatives are used for fixation, precautions should be taken with the material thus treated, as it may contain viable spores or other pathogens, which are destroyed after 24 hours of formalin treatment. Of the physicochemical methods tested involving microwaving, none was successful in eliminating viable spores from the test material.

Bacillus↗
PubMed1993

Tissue fixation methods alter the immunohistochemical demonstrability of neurofilament proteins, synaptophysin, and glial fibrillary acidic protein in human cerebellum.

This study has examined the effect of postmortem autolysis, type, and duration of fixation on neurofilament, synaptophysin, and glial fibrillary acidic protein (GFAP) antigen decay as demonstrated by immunohistochemistry, using a streptavidin-biotin peroxidase method. The system used consisted of 5 normal cerebellar cortices. Time intervals, temperature, mode of fixation and storage, and staining technique were well controlled. Anti-neurofilament antibodies comprised SMI-31, MNF, and BF-10 against phosphorylated epitopes, and SMI-32 against a non-phosphorylated epitope. Bouin's and B5 fixative, and Sensofix gave best results, whereas formaldehyde and paraformaldehyde fixation gave much lower immunoreactivity. Phosphorylated neurofilament epitopes were less affected by aldehydes than unphosphorylated epitopes. GFAP staining was most consistent after Bouin fixation while the monoclonal antibody was much more sensitive to the fixative used than the polyclonal one. Aspecific background staining increased considerably after a postmortem interval of 24 hours. Synaptophysin immunoreactivity, as demonstrated by SY-38, proved very sensitive to prolonged fixation and was of poor quality following formaldehyde and paraformaldehyde fixation. Knowledge of antigen decay due to postmortem artifacts is essential for the correct evaluation of immunoperoxidase studies of autolyzed tissues that have been fixed and stored in different modes and for variable time interval.

Antibodies↗
PubMed2003

[Methods of collagenous tissue fixation in the preparation of bioprostheses].

The use of biological materials in construction of bioprostheses requires the application of different chemical or physical procedures of fixation increasing bioprostheses resistance to enzymatic or chemical degradation and reducing their antigenicity. Methods typically concentrate on creating additional intra- and intermolecular chemical bonds between collagen molecules. This review focuses on the various methods of stabilization of collagenous tissues including chemical fixatives and physical agents.

Animals↗
PubMed2005

Influence of tissue fixation on the microextraction and identification of amyloid proteins.

In surgical pathology, correct immunohistochemical identification of AL amyloidosis poses a particular problem. Immunostaining for lambda- or kappa-light chains is commonly encountered even in non-immunoglobulin-derived amyloidoses, which leads to a false-positive classification as AL amyloidosis. In this respect, microextraction of amyloid proteins from surgical pathology specimens and their subsequent biochemical characterization may prove useful in reaching the correct diagnosis. In this study, we investigated systematically the influence of fixation on the extraction of amyloid proteins from amyloid-containing tissue samples. Tissue samples were obtained from a patient with generalized AA amyloidosis and from a second patient with generalized AL amyloidosis. The samples were stored either unfixed or fixed in phosphate buffered 4% p-formaldehyde, methacarn, or Bouin for 3 days, 1 week, or 1 month. Thereafter, proteins were extracted according to the procedure of Layfield et al, separated by SDS-PAGE and subjected to Western blotting, using antibodies directed against AA amyloid and immunoglobulin-derived lambda-light chain. Following this procedure, a variety of differently sized AA amyloid or lambda-light chain immunoreactive protein bands were found in both patients, which is typical for amyloid proteins. Fixation time did not per se prohibit the extraction of these amyloid proteins from tissue samples, which remained detectable irrespective of fixation time. Although all three fixatives impaired the resolution of some, but not all, individual amyloid proteins, this procedure may help to confirm or reject a diagnosis of AL amyloidosis, because detection of several lambda- or kappa-light chain immunoreactive protein bands in the low-molecular-weight range (<20 kDa) is a common characteristic of their amyloid nature.

Amyloid↗