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PubMed2005

Acetic acid-zinc-formalin: a safe alternative to B-5 fixative.

B5 fixation achieves superior morphologic detail. However, environmental concerns have led to labor-intensive and costly requirements for disposal of mercury-containing fixatives. We performed a blinded prospective study to find a safe, mercury-free alternative to B5. Morphology was evaluated with 6 fixatives, including B5, in a blinded fashion. Acetic acid-zinc-formalin (AZF) was selected for further evaluation of immunohistochemistry, in situ hybridization, and molecular analysis. AZF fixation resulted in overall staining and morphologic detail comparable to B5 and achieved equivalent or superior antigen preservation for immunohistochemical studies. Strong signal intensity was achieved with in situ hybridization, and DNA amplification could be successfully performed. AZF allows greater flexibility in fixation times, decreases decalcification time, and eliminates labor-intensive steps required for B5 processing.

Acetic Acid↗
PubMedDate not supplied

Permeafix: a useful tool to detect antigens and DNA in flow cytometry.

A new product called Permeafix has been developed for gentle fixation of cells and permeability of their membranes. This product is useful for the staining of intracellular antigens and parallel quantification of DNA content simultaneously with cell surface staining.

Antigens↗
PubMed1993

False aneuploidy in flow cytometric DNA analysis of paraffin embedded tissue: effects of Carnoy's fixation.

False aneuploidy was detected on flow cytometric DNA analysis of paraffin embedded axillary lymph nodes negative for tumor. It was hypothesized that "clearing" of axillary fat in Carnoy's solution to facilitate lymph node dissection might be responsible for false aneuploidy. Various tissues fixed overnight in Carnoy's were compared to formalin fixed paraffin embedded controls. Under these conditions no false aneuploid peaks were detected, but Carnoy's fixation did shift the G0/G1 histogram peak to the left, increase the G0/G1 CV and increase the S phase fraction relative to formalin fixed controls. It was then hypothesized that partial fixation of nodes in Carnoy's followed by formalin fixation might result in false aneuploid peaks. Twenty-two lymph nodes were partially fixed in Carnoy's for periods ranging from 5 to 60 min followed by complete fixation in formalin. Seven of these nodes did show false aneuploid peaks. By contrast, no aneuploidy was detected in formalin fixed controls. It was concluded that tissues in contact with Carnoy's solution may be a source of false aneuploidy and/or false elevation of S phase fraction. This reinforces the need for matched negative tissue controls for DNA analysis of paraffin embedded specimens whenever possible.

Acetates↗
PubMed1990

Does delay in fixation affect the number of mitotic figures in processed tissue?

The effect of delay in fixation on the number of mitotic figures in tissues has received little attention, and previous studies have reached differing conclusions. The numbers of mitotic figures in the normal mucosa of six colectomy specimens were counted with delays in fixation of 30 minutes, one hour, two hours, three hours and six hours for samples from each specimen. The numbers of mitotic figures were counted in 50 whole crypts in each specimen by two observers. All phases of mitosis were counted. The number of observable mitotic figures declined by about 30% with a delay in fixation of two hours and by 50% with a delay of six hours. This observation has important implications for the handling of surgical specimens.

Colon↗
PubMed1994

The influence of age of template DNA derived from archival tissue on the outcome of the polymerase chain reaction.

The analysis of DNA from archival tumour tissue for molecular alterations has been facilitated by the use of the polymerase chain reaction (PCR). Degradation of tissue prior to fixation and the nature of the fixative used influence successful amplification from archival tissue. Age of the archival tissue may also be a factor. To determine if this was so, DNA was extracted from 30 archival specimens of spleen spanning a 15 year period. Polymerase chain reaction was performed on all specimens using primers for exon 2 (307 bp) and exon 9 (1278 bp) of the hypoxanthine phosphoribosyl transferase (HPRT) gene. It was not possible to show that the age of archival tissue had an influence on the capacity to amplify exon 2 of the HPRT gene. It was not possible to amplify exon 9 of the HPRT gene from archival tissue.

DNA, Neoplasm↗
PubMed2002

Frozen-sectioning yields similar results as traditional methods for fetal cephalic examination in the rat.

BACKGROUND: An integral component of developmental toxicity studies is the evaluation of fetal anatomy, which consists of external, visceral, and skeletal examinations. The visceral examination includes an assessment of the fetal head which, in the rat, is typically completed after chemical fixation. Because chemical fixation requires approximately 7 days, a comprehensive visceral examination, including the head, of the fetal rodent cannot be completed at the time of cesarean section. An alternative method to chemical fixation was desired, whereby one could complete an overall visceral examination at the time of cesarean section. In addition, the method would also have to present fetal cranial structures in a manner consistent with that derived after chemical fixation. METHODS: Pregnant Sprague-Dawley rats were administered either 6-aminonicotinamide (6AN), 6 or 12 mg/kg on gestation day 13, or all-trans retinoic acid (RA) 5 or 25 mg/kg on gestation days 9 and 10, untreated dams served as a control group. On gestation day 20, fetuses were sacrificed and removed via cesarean section and one-half of the fetuses from each litter were placed in Bouin's solution (chemical fixation) and the remaining fetuses maintained under refrigeration until just before frozen tissue preparation (freeze fixation). Sectioning of the fetal head was similarly conducted after either chemical or frozen-fixation. Fetal cranial findings observed after chemical fixation were compared to those observed after frozen-fixation in the untreated control and 6AN and RA-treated groups. RESULTS: The incidence and severity of the cranial findings, including effects on the eye, brain, and palate, were similarly observed, regardless of fixation method. CONCLUSIONS: A frozen sectioning method for evaluation of the fetal head, yielding results comparable to those derived after chemical fixation, is described. This procedure provides a viable alternative to chemical fixation, and allows the teratologist to complete a comprehensive fetal visceral examination at the time of cesarean section.

6-Aminonicotinamide↗
PubMed2003

Detection of HER2/neu gene amplification in archival paraffin-embedded breast cancer tissues by fluorescence in situ hybridization.

We evaluated HER2/neu gene amplification by fluorescence in situ hybridization (FISH) in archival paraffin-embedded breast cancer tissues. Tumors from 63 human invasive breast cancers were categorized into two groups depending on whether the paraffin-embedded tissue blocks had been stored for more or less than 12 months duration. These were subjected to routine and modified FISH protocols. As microwave oven formalin fixation of tissues was carried out in the majority of the older archived specimens, the effect of this fixation method was also analyzed. FISH signals were obtained in all 13 archival specimens stored for less than 12 months. However, in 50 specimens stored for more than 12 months duration, the procedure was successful in only 10 specimens (20%), for which the pretreatment procedure had to be individually optimized for each specimen. There was no significant difference in the detection of FISH signals between microwave oven and routinely fixed specimens.

Breast Neoplasms↗
PubMed1989

Effect of fixatives on rat brain acetylcholinesterase activity.

Biochemical measurement of acetylcholinesterase (AChE) activity on sonicates of samples of rat cortex, striatum and cerebellum which had been treated with either 4% formaldehyde (HCHO) or 4% HCHO/0.5% glutaraldehyde suggest that fixation of tissue causes a rather large loss (50-80%) in AChE activity which may vary considerably from region to region of brain. Attempts to protect the activity by treatment with diisopropyl fluorophosphate (DFP) before fixation and reactivation after fixation with pyridine-2-aldoxime methiodide (PAM) were unsuccessful. The results suggest care must be exercised in attempting to use AChE histochemistry on fixed tissue for quantitative measurements.

Acetylcholinesterase↗
PubMed1994

A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues.

Immunohistochemistry is a powerful tool for tissue diagnosis and research. Although the frozen section has remained the gold standard for this important approach to evaluation of antigens in tissues, there is widespread acknowledgment of many limitations. Routine paraffin-embedded sections ware widely used for morphological examination of tissues but are not optimal for antigen preservation. In this study, paraffin-embedded tissues fixed with a simple buffer containing zinc as the primary fixative were compared with tissues fixed with routine formalin, zinc-formalin, paraformaldehyde, ethanol, a variety of commercial (non-formalin-containing) fixatives that have been recommended for reduced toxicity and improved antigen survival, and frozen sections. Human lymphoid tissues and a group of antibodies to antigens (CD1, CD4, CD7, CD8, CD19) usually preserved only in frozen tissue were used as a model system. Fixation in a simple solution of zinc acetate and zinc chloride in a Tris-Ca acetate buffer resulted in antigen preservation comparable to that in frozen sections with antibodies to these cell surface markers. Morphological preservation was comparable to formalin-fixed sections. The work presents a new method that represents the closest approach yet to a technique that combines optimal antigenic survival with the convenience and morphological preservation of traditional formalin-fixed tissue embedded in paraffin.

Acetates↗
PubMed1999

Limb reconstruction after high energy trauma.

Limb reconstruction techniques rely on stable external fixation to provide early limb function after major long bone injury. Bone may be generated by callus distraction techniques and internal techniques of moving bone segments used to fill bone defects. Soft tissue defects may be treated by acute shortening, although skin defects will also close spontaneously during bone transport as the leading edge of bone is covered with granulation tissue. External fixation is also used to cross joints permitting rest and repair of the joint. Hinges placed within the bars of the fixation frame may be used to correct deformities in the bone and soft tissue contractures using closed distraction techniques. These techniques are appropriate to metaphyseal fractures and diaphyseal fractures with bone loss. A major advantage is the lack of donor site morbidity, associated with skin flaps and large bone grafts. Acceptance of these techniques is growing whilst the methodology continues to improve. In more complicated cases, specialist training and dedicated hospital units with multidisciplinary support is desirable.

External Fixators↗
PubMed1996

Enhanced radiofrequency ablation of canine prostate utilizing a liquid conductor: the virtual electrode.

Conventional radiofrequency (RF) ablative techniques have shown promise for the treatment of symptomatic benign prostatic hyperplasia (BPH); however, present RF technology is limited by the small lesion size, necessitating several probe placements and heating cycles to achieve sizable lesions. This limitation is attributable primarily to a rapid increase in electrical impedance secondary to tissue desiccation and charring at the electrode tip. We devised a hollow screw-tip needle electrode that permits fixation to tissue, recording of temperature and impedance, infusion of fluid, and delivery of RF energy. Infusion of electrolyte solution (i.e., saline) into tissue prevents impedance rise by conducting RF energy away from the metal electrode and permits the creation of large lesions. By varying the conductivity of the perfusate (concentration and temperature), lesions of large diameter can be created in a controlled manner. To determine the long-term tissue effects, we applied this new modified RF technique to the prostates of five mongrel dogs in a chronic (0.5 to 8-week) study. The screw-tip electrode was serially embedded into each lobe of the perineally exposed glands with 1-minute infusion of 0.9% saline (2 mL/min) followed by application of RF energy (500 KHz, 50 W, 2-18 minutes) along with continuous saline infusion. Thermocouples were embedded 5 mm below and at the gland capsule, and RF application was discontinued when the temperature reached 50 degrees C at the periphery. Postoperatively, the animals were examined daily for clinical status and weekly for glandular changes using transrectal ultrasonography. At predetermined intervals, the animals were sacrificed and the prostates excised, measured, sectioned, and examined for histologic changes. Ablative tissue temperatures of 50 to 100 degrees C were produced while impedance remained stable. Four animals required a single catheterization for relief of urinary retention between days 2 and 3; otherwise, all animals demonstrated a quick and uneventful recovery with no edema detectable on day 7 ultrasound examination. The outside dimensions of the gland remained relatively constant throughout the study (+ or - 0.39 cm L + W + H). Histologic examination revealed coagulation necrosis (ablation) in both lobes of all prostates (69.94% + or - 16.62% of the gland) with tissueless cavities forming from the ablation area (28.71% + or - 8.24% of the gland) contained within the capsule surrounded by healthy tissue at the periphery. Intraprostatic lesions were obtained without any gross damage to surrounding tissue, including the bladder and rectal wall. Utilizing a liquid conductor in prostate tissue allows a single electrode-placement heating cycle for controlled ablation for the potential treatment of BPH. This new technique produces more extensive and uniform lesions than conventional RF procedures, and lesion size is limited only by the duration of RF energy application.

Animals↗
PubMed1978

A correlative chemical and histochemical study of the O-acetylated sialic acids of human colonic epithelial glycoproteins in formalin fixed paraffin embedded tissues.

Procedures are described for the isolation and identification of epithelial glycoproteins from formalin fixed, paraffin embedded specimens of human large intestine. The side chain O-acetylation patterns of the sialic acids of these glycoproteins were surprisingly similar to those of purified glycoproteins prepared from epithelial cells obtained from the same tissue before fixation. These results were consistent with those obtained by histochemical procedures performed on representative sections taken from the same tissue blocks. The methodology described permits a direct correlation of chemical and histochemical results obtained from the study of colonic epithelial glycoproteins of both normal and diseased tissues. It eliminates some of the difficulties associated with interpretation of the results by either discipline and may provide new information which would be unavailable by either chemistry or histochemistry alone.

Acetylation↗
PubMed2002

Combination of fixation using PLP fixative and embedding in paraffin by the AMeX method is useful for histochemical studies in assessment of immunotoxicity.

To establish a method for processing lymphoid organs suited to morphological, immunohistochemical and enzyme histochemical analyses for assessment of immunotoxicity, we examined a combination of fixation with periodate-lysine-paraformaldehyde (PLP) fixative and embedding in paraffin by the AMeX method (PLP-AMeX method). Spleen and thymus removed from monkeys and rats were fixed in PLP fixative for 6 hours at 4 degrees C. After fixation, specimens were processed and embedded in paraffin by the AMeX method. In hematoxylin and eosin-stained sections, tissue architecture was well preserved. In immunohistochemical staining, markers of T lymphocytes (CD3, CD4, CD8), B lymphocytes (monkey: CD20cy, rat: CD45RA) and macrophage (monkey; CD68, rat: ED-1) were well identified according to their specificities, although the staining intensity of CD8 in the monkey and CD4 in the rat were somewhat weaker in PLP-AMeX-prepared sections than in those frozen. In enzyme histochemical staining, alkaline phosphatase activity was well preserved in neutrophils. In toluidine blue- and Giemsa-stained sections, eosinophil granules and the metachromasia of granules in basophil/mast cells were clearly detectable. These findings suggest that the PLP-AMeX method is a powerful tool for assessment of immunotoxicity.

Animals↗
PubMed1993

The ultrastructure of the connective tissue matrix of skin and cartilage after high-pressure freezing and freeze-substitution.

Studies designed to investigate the ultrastructure of the connective tissue matrix have historically relied on chemical fixatives to stabilize tissue microarchitecture. However, conventional fixatives are not completely effective in retaining many matrix constituents, including proteoglycans. Fixative recipes have been modified to include agents that retain proteoglycans, but they precipitate the glycosaminoglycan moiety into electron-dense granules and therefore do not preserve native microstructure. To avoid the structural artifacts introduced by aqueous fixatives, we prepared cartilage and skin by a cryostabilization procedure that included high-pressure freezing and freeze-substitution. Although similar approaches have been applied previously for study of connective tissue, our results and interpretation of matrix structure are significantly dissimilar. In optimally preserved areas of cartilage, collagen fibrils are continually surrounded by a densely staining sol. Empty fluid spaces are absent. In less optimally preserved areas, artifacts are noted and described, including a network that mimics the expected structure of proteoglycan. Similarly, the dermal matrix of human skin contains a preponderance of densely staining material that almost fills the voids commonly seen after aqueous fixation. Decorin, immunolocalized to the surface of dermal collagen fibrils, appears to be retained after this procedure.

Animals↗