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PubMed2002

Evaluation of HER-2/neu immunohistochemical assay sensitivity and scoring on formalin-fixed and paraffin-processed cell lines and breast tumors: a comparative study involving results from laboratories in 21 countries.

Variation in assay sensitivity was studied in more than 90 laboratories that assayed 4formalin-fixed, paraffin-processed breast and ovarian carcinoma cell lines with graded levels of HER-2/neu protein overexpression and known levels of HER-2/neu gene amplification, in addition to breast carcinomas fixed and processed in the laboratories. Main methods were the HercepTest (DAKO, Ely, England) and individualized protocols using a polyclonal antibody and the CB11 clone. While the proportion of laboratories achieving appropriate results with the HercepTest was significantly higher than for participants using other assays, laboratories using other assays showed significant improvement in the second assessment run. The level of agreement in evaluations by 26 laboratories using the HercepTest was excellent on cell lines and tumors and was significantly greater than that achieved by the remaining 41 laboratories using other immunohistochemical methods. While laboratories using the DAKO HercepTest had the highest level of reproducibility in assay sensitivity and evaluation, the significant improvement in results by laboratories using other antibodies in the second assessment run suggests that stringent quality control and an ongoing quality assurance program using a standard reference material have the potential to improve the reliability of immunohistochemical assays for HER-2/neu, regardless of the antibody used.

Breast Neoplasms↗
PubMed1995

Fixation of whole eyes: the role of fixative osmolarity in the production of tissue artifact.

BACKGROUND: Whole eyes fixed in 4% buffered formaldehyde (10% neutral buffered formalin) demonstrate a variety of artifacts, including separation of the neurosensory retina from the retinal pigment epithelium. We postulate that the osmolarity of 4% buffered formaldehyde causes contraction of the internal compartments of the eye leading to several artifactual changes commonly observed in routine histologic sections. METHODS: In part I of the study, enucleated animal eyes were examined histologically after immersion in different concentrations of formaldehyde. The variables of fixation and processing were kept constant except for the concentration and osmolarity of formaldehyde. In part II, enucleated animal eyes were used to empirically determine the optimal mixture of formaldehyde and glutaraldehyde for fixation based on subjective assessment of histologic sections. RESULTS: In the first part of the study, the post-fixed volume of the anterior chamber and vitreous increased as the concentration (and osmolarity) of formaldehyde decreased. In part II of the study, fixation of whole eyes was optimal with a mixture of 1% buffered formaldehyde and 1.25% glutaraldehyde. The neurosensory retina was less likely to detach from the retinal pigment epithelium, and the anterior chamber retained a more normal shape with this fixative. CONCLUSIONS: Volume contraction of whole eyes fixed in 4% buffered formaldehyde is caused by the relatively high osmolarity of the fixative. Immersion fixation of whole eyes for 36 h (or longer) in 1% buffered formaldehyde/1.25% glutaraldehyde reduces tissue distortion without compromising cellular preservation.

Animals↗
PubMed1992

Delay in fixation does not affect the immunoreactivity of proliferating cell nuclear antigen (PCNA).

The effect of delayed fixation on the immunoreactivity of proliferating cell nuclear antigen (PCNA) was investigated using eight breast carcinomas. Topologically shuffled samples of each tumour were immersed in fixative at times of 0.5, 1, 2, 4, 6, 18, and 24 h after surgical removal. In addition to a PCNA index (percentage of positive cells per 1200 tumour cells), a semi-quantitative PCNA grading system was used, based on estimates of more than or less than 50 per cent of positive tumour cells at each time interval. The PCNA index of six tumours increased by a mean of 10 per cent with a fixation delay of 24 h. The PCNA grade of all eight tumours showed no change with delayed fixation.

Antigens, Neoplasm↗
PubMed1993

Improvements in the silver-staining technique for nucleolar organizer regions (AgNOR).

The silver-staining technique for nucleolar organizer regions (AgNOR) of Ploton et al., as popularized by Crocker, is being widely used for evaluation of nucleolar function, especially in neoplasia. It suffers from limited reliability, background staining, precipitates, and fading of the sections. Factors were identified that affect these problems. The oxidation-reduction level and gelatin used are particularly important. An improved procedure is presented which incorporates pre-reduction of the sections, selection of an optimal gelatin, and post-treatment of the sections to produce a permanent preparation. It is compatible with many fixatives and with other stains used before or after the silver stain.

Chemical Precipitation↗
PubMed1999

The serous demilune of rat sublingual gland is an artificial structure produced by conventional fixation.

The ultrastructure of the secretory end-piece of the rat sublingual gland was examined in samples prepared by rapid freezing and freeze-substitution method, and results were analyzed in combination with 3-D images reconstructed by computer graphics from light micrographs of serial sections. Fixation by rapid freezing followed by freeze-substitution preserved cellular ultrastructures, especially the membrane structure, in perfect condition, and demonstrated the terminal portion of the sublingual gland to be a compound branched tubulo-alveolar gland with serous cells distributed throughout the end-pieces. All the serous cells aligned with mucous cells to surround a common lumen, leaving no demilune structure. In contrast, samples fixed by the conventional immersion method showed distended mucous cells displacing the serous cells toward the basal portion of the acinus to form the demilune structure. The luminal space was also compressed and appeared disconnected from the serous cells. From these observations, the serous demilune that for more than 130 years has been believed to be an actual histological entity was proved to be an artificial structure produced through compression by the hydrated and expanded mucous cells during immersion fixation.

5'-Nucleotidase↗
PubMedDate not supplied

Microwave fixation provides excellent preservation of tissue, cells and antigens for light and electron microscopy.

It was demonstrated that microwave energy used simultaneously in combination with low concentrations of glutaraldehyde (0.05%) and formaldehyde (2.0%) rapidly preserved light microscopic histology and excellent fine structural details, as well as a variety of cytoplasmic and membrane-bound antigens. Specimen blocks up to 1 cm3 can be fixed in as brief a time as 26 ms using a specially designed microwave device (ultrafast microwave fixation method). The fast microwave fixation method, using a commercially available device, was successfully used to preserve granule-bound rat mast cell chymase which was subsequently detected by a postembedding immunogold procedure. Control of the following parameters is important to the microwave fixation method: (1) specimens with one dimension less than 1 cm; (2) irradiation temperatures lower than 50 degrees C; (3) irradiation times less than 50 s; (4) immediate replacement of the postirradiation solution with cold storage buffer; (5) fixing the specimen within 15 min after it is removed from its blood supply.

Animals↗
PubMed1989

Paraffin sections of nervous tissue supravitally stained with methylene blue: a new, reliable and simple fixation technique.

Two steps are recommended for fixation of central nervous tissue after supravital staining with methylene blue: application of ammonium heptamolybdate followed by a paraformaldehyde-glutaraldehyde mixture. The two fixatives complement each other so that both dye and tissue are optimally fixed. Molybdate renders the dye water and alcohol insoluble, the aldehyde mixture conditions the tissue for embedding in paraffin. This technique is a suitable alternative to conventional whole mount and frozen sectioning methods.

Animals↗
PubMed2005

Dual cryogenic fixation for mechanical testing of soft musculoskeletal tissues.

Mechanical testing of soft musculoskeletal structures like tendons and ligaments are essential to medical advances. A long-standing limitation for testing these structures in isolation has been the ability to solidly fix both ends of the tendon. Cryogenic fixation technology was leveraged into the development of a dual cryogenic fixation (DCF) device. Results of the study show that the DCF allows tendons to be tested in isolation, at physiologic temperatures, with excellent reproducibility.

Animals↗
PubMed2006

Immunocytochemical analysis of D-serine distribution in the mammalian brain reveals novel anatomical compartmentalizations in glia and neurons.

D-Serine is a co-agonist at the NMDA receptor glycine-binding site. Early studies have emphasized a glial localization for D-serine. However the nature of the glial cells has not been fully resolved, because previous D-serine antibodies needed glutaraldehyde-fixation, precluding co-localization with fixation-sensitive antigens. We have raised a new D-serine antibody optimized for formaldehyde-fixation. Light and electron microscopic observations indicated that D-serine was concentrated into vesicle-like compartments in astrocytes and radial glial cells, rather than being distributed uniformly in the cytoplasm. In aged animals, patches of cortex and hippocampus were devoid of immunolabeling for D-serine, suggesting that impaired glial modulation of forebrain glutamatergic signaling might occur. Dual immunofluorescence labeling for glutamate and D-serine revealed D-serine in a subset of glutamatergic neurons, particularly in brainstem regions and in the olfactory bulbs. Microglia also contain D-serine. We suggest that some D-serine may be derived from the periphery. Collectively, our data suggest that the cellular compartmentation and distribution of D-serine may be more complex and extensive than previously thought and may have significant implications for our understanding of the role of D-serine in disease states including hypoxia and schizophrenia.

Aging↗
PubMed2006

Cytogenetic features of ependymoblastomas.

Ependymoblastomas are very rare and highly malignant embryonal tumours of the central nervous system with distinctive multilayered rosettes being the main histopathological feature. They are a diagnostically challenging subtype of embryonal tumours, whose genetic features are unknown. Primary ependymoblastomas from four children (one boy, three girls; mean age 24.8 months, range 4-41 months) were investigated by comparative genomic hybridisation (CGH), to our knowledge constituting the only cohort of this entity studied by cytogenetic means. DNA copy number changes were found in each case, consisting mainly of gains of chromosome 2 as well as losses of chromosomes 6q and 13q (75% each). The tumours showed between one and five aberrations with a mean of 3.25 DNA copy number changes per case, with gains being less frequent than losses (1.25 gains vs 2 losses per case). The youngest patient showed the least imbalances (one), whereas the oldest child presented with the most aberrations (five). Clinical follow-up data were available for three of the four patients. All three had died of their disease after a post-operative survival of 9 months (range 6-14 months). Our CGH data suggest that ependymoblastomas show distinct and fairly consistent chromosomal aberrations.

Brain Neoplasms↗
PubMed1993

Effect of calcification and formalin fixation on in vitro distensibility of human femoral arteries.

To determine the effect of calcification on the distensibility of human arteries, we measured changes in lumen cross-sectional area as a function of pressure in excised segments of calcified and noncalcified human femoral arteries with the use of intravascular ultrasound. Cross-sectional area at zero pressure was greater in calcified artery segments (22.1 +/- 4.4 mm2) compared with noncalcified arteries (18.9 +/- 2.4 mm2). Calcified arteries were significantly less distensible than noncalcified arteries (2.5-fold). To assess the validity of mechanical tests of fixed arteries, pressure-area relations were also obtained after formalin fixation: mean unstressed cross-sectional area decreased significantly in noncalcified arteries (24%) but did not change significantly in calcified arteries. Formalin fixation decreased distensibility significantly (68%) in noncalcified arteries but not in calcified arteries. These results indicate that decreased lumen size and increased rigidity are induced by formalin fixation in noncalcified arteries but not in calcified arteries, and that this is attributable to the lower distensibility of calcified arteries before fixation. Because histologic examination is commonly used to evaluate severity of lumen narrowing in studies of atherosclerosis, this differential effect of formalin fixation may cause calcified arteries to appear less severely obstructed than noncalcified arteries.

Calcinosis↗
PubMed1997

The rate of aldehyde fixation of the exocytotic machinery in cultured hippocampal synapses.

The rate at which the transmitter release machinery is fixed by 2% glutaraldehyde at hippocampal synapses and the amount of release evoked by the fixative have been investigated. We recorded from hippocampal cells while fixative was applied with a rapid flow system. Release is blocked in less than a second and fixative-produced exocytosis is at most a few percent of what would be caused by a hypertonic stimulus that completely depletes the readily releasable pool of vesicles. The postsynaptic receptors for glutamate cease to respond to agonist with a time constant of approximately 3 s when fixative is applied. We conclude that some essential component of the exocytotic apparatus is fixed in less than a second and that the fixative does not significantly deplete the readily releasable pool.

Aldehydes↗
PubMed2003

SEM observation of the insect prepared by microwave irradiation.

The ptilinum of the fly and the compound eye are among the most fragile organs encountered during conventional procedures of morphological sample fixation. In order to identify a fixative suitable for preparing such samples for scanning electron microscopy, we examined various fixation conditions using microwave irradiation (MWI). The conditions examined were: (i) fixatives; (ii) temperature; (iii) concentration; (iv) duration; (v) dehydration; and (vi) substitution. The identified optimal conditions were 5% glutaraldehyde with MWI (350 W, 5 min). The MWI was continued until the maximal temperature of 75 degrees C was attained, followed by intermittent irradiation to maintain a temperature of 75 degrees C. After irradiation, the sample was left at room temperature for 24 h in the fixative and then dehydrated in increasing concentrations of ethanol. Each step in the ethanol series lasted for 24 h. The final absolute ethanol step included three solution changes, with each incubation lasting 1 h. A subsequent stepwise substitution of t-butyl alcohol for ethanol was conducted by reducing the ratio of 100% ethanol to t-butyl alcohol from 2:1 to 1:1 and then 1:2 (24 h each). The substitution was completed by three solution changes using 100% t-butyl alcohol, 30 min each. The best results were obtained by freeze-drying samples using t-butyl alcohol. The use of MWI improved fixative permeation, which occurred at a uniform rate throughout the sample. Comparison with temperature in a water bath at 75 degrees C indicated that the fixation effect of MWI was due to its heat generation in addition to some unknown mechanism.

Animals↗
PubMed1993

Histochemical heterogeneity of mast cells in rat dermis.

The dermal mast cells of Wistar rats were studied following fixation in either 10% phosphate buffered formalin or Carnoy's solution and staining with either toluidine blue or alcian blue:safranin O. Granules of mast cells appeared heterogeneous following fixation with formalin and staining with alcian blue:safranin O, but not when stained with toluidine blue. The number of mast cells observed in skin fixed in Carnoy's solution was greater than the number observed in equivalent samples of skin fixed in formalin (p < 0.01) when both samples were stained with toluidine blue. In formalin fixed skin stained with alcian blue:safranin O, there were three populations of mast cells designated as "blue," "red" or "mixed." "Blue" mast cells, containing only alcian blue stained granules, "red" mast cells, containing only safranin O stained granules, and "mixed" mast cells, containing both alcian blue and safranin O stained granules accounted for 77.6 +/- 3.0, 6.6 +/- 2.5 and 15.8 +/- 2.5% of the total mast cell population, respectively. In skin specimens fixed in Carnoy's solution and stained with alcian blue:safranin O the mast cells contained only blue granules (alcian blue positive). The number of mast cells observed in Carnoy's fixed skin sections was less than the number seen in formalin fixed skin sections when both were stained with alcian blue/safranin O. This indicates that there is a group of mast cells which do not stain with safranin O after fixation with Carnoy's solution.

Animals↗
PubMed1980

Carbon dioxide fixation and related properties in sections of the developing green maize leaf.

Light and dark (14)CO(2) assimilation, pulse-chase ((14)CO(2) followed by (12)CO(2)) labeling experiments both in the light and in the dark, photorespiratory activity and some enzymes (ribulose 1,5-bisphosphate (RuBP) carboxylase, phosphoenolpyruvate (PEP) carboxylase, and NADP-malic enzyme) were followed in sections of 2.5 centimeters from the base (younger tissue) to the tip (oldest tissue) of the green maize leaf. Tissue was taken from the third leaf of 12- to 16-day-old plants consisting of sections 0 to 2.5 centimeters (base), 4.5 to 7.0 centimeters (center) and 9.0 to 11.5 centimeters (top) measured from the base. Some of these properties were also determined in the intact leaves of 4-day-old maize plants.Electron microscopy indicated a Kranz anatomy in all sections. Differentiation into mesophyll granal chloroplasts and bundle sheath agranal chloroplasts had taken place only in the center and top pieces.All of the sections contained PEP carboxylase, RuBP carboxylase, and NADP-malic enzyme. The ratio of PEP:RuBP carboxylase increased from 3.03 (top) to 4.66 (base) whereas the PEP carboxylase:NADP-malic enzyme ratio rose from 2.87 (top) to 9.57 (base).Under conditions of light or dark, the majority of the newly incorporated (14)CO(2) was found in malate and aspartate in all sections and in 4-day-old leaves. The (14)C-labeling pattern typical of C(4) plants was present in the center and top sections and to a lesser extent in the 4-day-old leaves. In the base tissue, the percentage of radioactivity in malate and aspartate remained relatively constant both during photosynthesis and pulse-chase experiments. In contrast, radioactivity in glycerate-3-phosphate decreased with time coupled to an increase in sugar phosphates. To account for the isotopic pattern in the base tissue, parallel fixation by PEP carboxylase and RuBP carboxylase was proposed with the photosynthetic carbon reduction cycle functioning to some extent independently within the bundle sheath chloroplasts. The apparent lack of cooperation between the mesophyll and bundle sheath cells may have been due to inadequate levels of NADP-malic enzyme required for shuttling carbon as CO(2) from the PEP carboxylase products to the Calvin cycle.

Journal Article↗