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PubMed1991

Influence of fixation and immunohistological technique on accuracy, precision and inter-observer reproducibility of plasma cell counting.

Recently two highly sensitive and specific diagnostic criteria for Sjögren's syndrome based on percentages of IgA-, IgG-, and IgM-containing plasma cells measured in immunohistologically stained labial salivary gland tissue have been described. The reliability of such a criterion is dependent on the accuracy, precision and inter-observer reproducibility in plasma cell counting. The present study evaluates the effect of tissue fixation and immunohistological procedures on the aforementioned factors. Immunoglobulin (Ig)-containing plasma cells in sections of lamellated submandibular salivary gland tissue, alternately fixed in a 4% buffered formol solution or formol-sublimate solution and stained with an indirect immunoperoxidase and unlabelled peroxidase anti-peroxidase (PAP) method respectively, were enumerated by three independent observers. Relative numbers of Ig-containing plasma cells appeared to be less sensitive for systematic errors due to tissue fixation and immunohistological procedure than absolute numbers of Ig-containing plasma cells. The best inter-observer reproducibility of plasma cell counts was obtained in sections from formol sublimate-fixed specimens stained according to the PAP procedure.

Analysis of Variance↗
PubMed1994

The natural history of alloplastic implants in orbital floor reconstruction: an animal model.

We developed a new animal model to recreate the condition of an open fracture in communication with the maxillary sinus. We then studied wound healing of the sinus wall structures following fracture in the presence of an alloplastic implant. This model is designed to simulate the alloplastic repair of an orbital floor fracture in humans. The New Zealand White rabbit was used as the animal model. Standardized 8-mm defects were made bilaterally in the maxillary sinuses to include bone and mucosa in 21 rabbits. Two different implants were placed in the soft-tissue pockets to obturate the defects, exposing one surface of the implant to the open sinus. Medpor porous polyethylene and silicone implants were compared. Animals were killed at 1, 2, 3, and 4 weeks and at 2, 4, and 5 months after implantation. Gross examination of the specimens for the amount of mucosal closure and implant tissue fixation was performed. Histological sections were evaluated for bone and soft-tissue morphology juxtaposed to the implant. Complete closure of the mucosal defect was demonstrated with both types of implants. Medpor implants showed both vascular and soft-tissue ingrowth into its pores by week 1. Bone ingrowth was seen by week 3. Closure of the Medpor obturated defects occurred more rapidly than in the silicone group (p < 0.004 at week 4). The Medpor implants demonstrated bone and soft-tissue fixation, and mature overlying mucosa was reconstituted over the defects. Silicone implants demonstrated a fibrous tissue reaction within 1 week of implantation and they never became fixed to bone or soft tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Immunohistochemical localization of ANG II AT1 receptor in adult rat kidney using a monoclonal antibody.

Molecular and functional studies have suggested that AT1 receptors are present in most nephron segments, yet direct demonstration of AT1 at these sites is lacking. The present study was performed to determine the intrarenal localization of the AT1 receptor utilizing a monoclonal anti-peptide (amino acid residues 8-17) antibody (6313/G2) in adult male Sprague-Dawley rats. Western blot analysis of kidney protein extracts showed a predominant 41-kDa immunoreactive band corresponding to the molecular weight of the deduced cDNA sequence. To determine optimal fixation conditions, kidney tissues were immersion fixed in Bouin's solution, 10% buffered Formalin, or 4% paraformaldehyde. Specificity of immunostaining was documented by preadsorption of the antibody with the immunogenic peptide sequence. Prominent AT1 immunostaining was visualized in the proximal tubule brush-border and basolateral membranes. In addition, distal tubules, cortical and medullary collecting ducts, and the renal arterial vasculature exhibited specific immunoreactivity. Glomerular staining for AT1 was observed in mesangial cells and podocytes. Macula densa cells stained positively. Similar localization of the AT1 receptor was obtained using the three tissue fixation methods, although the intensity of vascular and glomerular staining was highest in Bouin-fixed tissues. The present study demonstrates that the AT1 receptor is more widely distributed along the nephron than previously described and includes renal vascular smooth muscle and proximal and distal epithelial sites.

Amino Acid Sequence↗
PubMed2001

Immunohistochemical localization of zona pellucida proteins ZPA, ZPB and ZPC in human, cynomolgus monkey and mouse ovaries.

The zona pellucida of mammalian oocytes plays an important role in binding and activation of sperm cells during the molecular events leading to fertilization. The genes coding for the three zona pellucida glycoproteins ZPA, ZPB, and ZPC of various species including mouse, dog, and human have been cloned and sequenced by several groups. However, it has remained a matter of debate as to whether the oocytes alone or in conjunction with the surrounding granulosa cells express and deposit these proteins to form the zona pellucida matrix. Addressing this unresolved issue, we assessed the expression and localization of all three zona pellucida proteins in ovaries of human, cynomolgus monkey and mice using immunohistochemical methods. In addition, oocyte-specific expression of ZPC from the primordial stage onward was confirmed by in situ hybridization. In sections of human ovaries, ZPA, ZPB, and ZPC proteins were immunohistochemically detected in the cytoplasms of primordial oocytes and during later stages of folliculogenesis in the zona pellucida matrices of oocytes. In sections fixed with formalin, a clear homogeneous ring was visible around the oocyte and no staining of granulosa cells was observed. In contrast, staining of ZP proteins was also observed between granulosa cells when Bouin's reagent had been used for tissue fixation. Thus, the original zona pellucida architecture was better preserved by formalin fixation. We further demonstrated that dissolution of the zona pellucida of isolated bovine oocytes occurred after they were exposed to Bouin's reagent. In summary, these results demonstrate that in mice, monkeys and humans, zona proteins are expressed and assembled exclusively by the oocyte and not by the granulosa cells. Previously observed results of ZP expression by an involvement of granulosa cells might therefore be the result of an improper fixation of the tissues leading to the disruption of the zona pellucida. Additionally this study highlights the importance of choosing the correct fixative for immunohistochemistry, not only for the usual reason of retaining antigenicity, but rather to retain the entire architectural structure.

Acetic Acid↗
PubMed2001

AmpFlSTR Profiler Plus and AmpFlSTR COfiler analysis of tissues stored in GenoFix, a new tissue preservation solution for mass disaster DNA identification.

A preliminary study was conducted to assess the capability of a new alcohol-based tissue fixative, GenoFix, to preserve DNA from biopsy tissues stored at room temperature and/or -20 degrees C in a freezer, for subsequent short tandem repeat (STR) DNA typing analysis. Fresh human smooth muscle samples were stored at room temperature in GenoFix for one month and up to one year and seven months before being processed using the megaplex STR systems, AmpFlSTR Profiler Plus and AmpFlSTR COfiler. Alternatively, muscle tissues in GenoFix were placed at -20 degrees C in a freezer for up to 3 1/2 years following two to three months in the fixative at room temperature. DNA analysis was also carried out on tissues stored in GenoFix for one month at room temperature and subsequently paraffin-embedded and stored at room temperature for four years. The AmpFlSTR Profiler Plus and AmpFlSTR COfiler STR profiles produced, using DNA extracted from all fixed tissue samples, were of very good quality. The fluorescent signals were well balanced across the nine STR loci or six loci comprised in the megaplexes surveyed and profiles showed no differences with those observed for the control blood of the respective donor patients. Continuous exposure to GenoFix at room temperature (up to one year and seven months) did not compromise the STR typing analysis of the fixed tissues. No adverse effects were noted on the STR typeability of tissues fixed with GenoFix and stored at -20 degrees C in a freezer for up to 3 1/2 years. STR profiles generated from the paraffin-embedded tissues fixed in GenoFix were of excellent quality. This preliminary study suggests that GenoFix can be used to store tissue samples at room temperature for up to one year and seven months or at -20 degrees C in a freezer for longer storage (up to 3 1/2 years). This new and odorless tissue fixative promotes tissue and DNA preservation in a very effective manner and as such may prove useful in criminal investigations or mass disaster identifications carried out in remote locations and in which a small or large number of tissue samples are collected for further analyses.

Biopsy↗
PubMed1983

Preclinical and clinical evaluation of topical acid products for skin tumors.

Distinct differences between topical acid preparations can be detected by simple preclinical and clinical pharmacologic models designed to evaluate potential utility in the treatment of superficial skin tumors. The models distinguish a tissue-dissolving (ulcerating) effect from a tissue-fixation (mummifying) effect, as reflected by a distinct and lasting discoloration of hair in vitro or the skin surface in vivo. Double-blind clinical comparison of the response to different preparations of matched pairs of small superficial skin tumors in 33 subjects demonstrated for greater utility for nitric acid preparations of appropriate strength (6 to 7N), with added oxidizable organic acids, over the mineral acid alone. The nitrate reduction products generated in such preparations markedly speed up discoloration in the models and apparently contribute to the improved clinical utility of the topical treatment by enhancing the speed and intensity of tissue fixation, but not tissue erosion. The architecture of lesions eradicated by such mixtures is generally adequately preserved for histologic diagnosis of the extruded tissue.

Administration, Topical↗
PubMed1996

Fluorescence techniques for diagnosing intestinal microsporidiosis in stool, enteric fluid, and biopsy specimens from acquired immunodeficiency syndrome patients with chronic diarrhea.

OBJECTIVE: To evaluate three fluorescent chitin stains for detecting microsporidia spores in specimens from acquired immunodeficiency syndrome (AIDS) patients with chronic diarrhea. METHODS: We compared the Fungifluor, Calcofluor White, and Fungiqual A fluorochrome stains for identifying Enterocytozoon bieneusi and Septata intestinalis spores in stool, intestinal fluid, biopsy imprints, and paraffin biopsy sections. The modified chromotrope trichrome stain was used as the standard light microscopic technique for stool and fluid specimens. Stained and unstained paraffin sections and fluid preparations were also evaluated. Multiple specimens from 50 consecutive symptomatic AIDS patients and archival material from known microsporidia-positive AIDS patients were analyzed. RESULTS: Spores of E bieneusi and S intestinalis fluoresce brightly with all three fluorochrome stains in all of the types of diagnostic specimens. Fluorescing debris and the much larger fungal forms were readily distinguished. Spores were equally well detected in unfixed and formalin-fixed stool specimens, but were not as well detected after sodium acetate-acetic acid, polyvinyl acetate, and ethanol fixation. Bouin's tissue fixative gave a higher background staining than formalin. Spores were readily detected in archival paraffin sections and stool preparations, even when the specimens had been stained previously. Repeat fluorochrome staining was possible. The methods also could detect extraintestinal parasites in paraffin sections. CONCLUSION: The three fluorescent chitin stains are sensitive and rapid methods for detecting microsporidia spores in stool, intestinal fluid, biopsy imprint, and tissue specimens, even from archived material.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Enzyme histochemistry and immunohistochemistry with freeze-dried or freeze-substituted resin-embedded tissue.

Freeze-drying or freeze-substitution, combined with low-temperature resin-embedding, represents a new approach to the optimum preservation of tissue for enzyme histochemistry and immunohistochemistry. This method, which avoids tissue fixation, combines excellent tissue morphology with the preservation of enzyme activity and immunoreactivity and allows high-resolution enzyme histochemical and immunohistochemical studies to be performed. The activity of a wide range of enzymes can be demonstrated in sections of freeze-dried or freeze-substituted resin-embedded tissue. Enzymes are retained in situ with high activity, accurate localization and no diffusion. Immunohistochemical studies can also be performed on resin sections, and antigens--especially labile antigens--are immobilized in situ without denaturation and can be demonstrated with high sensitivity and accurately localized. This method allows the localization and distribution of enzymes and antigens to be studied in relation to excellent histological and cytological detail.

Animals↗
PubMed1988

Technical considerations in the immunohistochemical demonstration of intermediate filaments.

The intermediate filaments represent an important group of differentiation markers that have proven to be useful for the diagnosis and classification of human neoplasms. The immunohistochemical demonstration of these proteins is subject to numerous technical variables that include specificities and patterns of reactivities of polyclonal antisera and monoclonal antibodies, cross-reactivities of "specific" monoclonal antibodies with other intermediate filaments and other classes of antigens, masking of certain epitopes due to physiologic or pathologic changes in cells, effects of tissue fixation and subsequent tissue processing, and the sensitivity of the particular immunohistochemical detection system employed. Moreover, a wide variety of microenvironmental signals may affect the patterns of expression of intermediate filament proteins in different pathological conditions. For accurate interpretation of intermediate filament immunoreactivity, the pathologist must be familiar with the various sources of error relating to false positive or negative results. This requires the availability of optimally fixed tissues, a well-characterized set of antibodies to intermediate filament antigens and access to the results of extensive performance testing.

Antibodies, Monoclonal↗
PubMed1956

Quantitative estimation of lyo- and desmoenzymes in tissue sections with and without fixation.

1. Tissue sections eight microns thick were exposed to various experimental conditions used in histochemistry, and the effect upon the activities of esterase, the phosphatases, leucine aminopeptidase, beta-glucuronidase, and arylsulfatase was determined colorimetrically. 2. Significant differences were found in the amounts of the lyo and desmo fractions of these enzymes. The desmo components were found to be for esterase, alkaline phosphatase, leucine aminopeptidase, acid phosphatase, beta-glucuronidase, and arylsulfatase, (1/3), 2/3, 2/3, (1/2), (1/8), and (1/8) of the total enzymatic activity respectively. 3. Variations in the time and in the temperature at which diffusion was studied and of the pH and salt concentration of the solution into which the sections were placed, resulted in differences in the amount of enzymatic activity which remained in the tissue section. Some enzyme loss by diffusion was noted even after fixation of the tissue section. 4. The significance of the findings with respect to some of the concepts of localization of enzymes in tissue sections was discussed.

Acid Phosphatase↗
PubMed1985

Postinflammatory ossicular fixation: CT analysis with surgical correlation.

Postinflammatory ossicular fixation is a common problem encountered by the otologic surgeon upon exploration because of conductive hearing loss in patients with chronic otitis media. These nonotosclerotic noncongenital lesions take three pathologic forms: fibrous tissue fixation (chronic adhesive otitis media), hyalinization of collagen (tympanosclerosis), and new bone formation (fibro-osseous sclerosis). Fibrous tissue fixation appears on CT as nonbony, noncalcific soft-tissue debris encasing some or all of the ossicular chain. Tympanosclerosis appears as unifocal or multifocal punctate or weblike calcifications in the middle ear cavity or on the tympanic membrane. This debris may be in direct apposition to the ossicular chain or may replace the suspensory ligaments in symptomatic patients. New bone formation has been identified only in the attic and is the least common manifestation. Thick bony webs or generalized bony encasement may be present at CT. More than 300 patients with the clinical diagnosis of chronic otitis media have been examined. This study encompasses 23 proved cases.

Adult↗
PubMed2001

Arthroscopic Bankart repair with the Suretac device for traumatic anterior shoulder instability in athletes.

Arthroscopic treatment of anterior shoulder instability in the athlete has evolved tremendously over the past decade. Currently, most techniques include the use of suture and suture anchors. However, the variety of arthroscopic instruments and techniques that are available shows the complexity of intra-articular tissue fixation, which includes anchor placement, suture passing, and knot tying. Stabilization using the Suretac device (Acufex Microsurgical, Mansfield, MA) simplifies tissue fixation by eliminating the need for arthroscopic suture passing and intra-articular knot tying. However, a successful outcome is highly dependent on accurate patient selection. Preoperative evaluation, examination under anesthesia, and the pathoanatomy defined by a thorough arthroscopic examination suggest the most effective treatment strategy. The ideal candidate for shoulder stabilization using the Suretac device is an athlete with a relatively pure traumatic anterior instability pattern with detachment pathology (e.g., Bankart lesion) and minimal capsular deformation.

Absorbable Implants↗
PubMed2007

Behavior of the different implant materials in acute infection and efficacy of antibiotherapy: experimental study in rats.

In this study, we propose a comparison of the behaviors of four different implant materials in case of acute infection: expanded polytetrafluoroethylene (e-PTFE), porous high density polyethylene (PHDPE), silicone, and autogenous cartilage tissue. The efficacy of prophylactic and therapeutic antibiotic therapies was also investigated in a rat model as four groups: group A, acute infection and no antibiotic therapy (n = 24); group B, acute infection and prophylactic antibiotic therapy (n = 24); group C, acute infection and therapeutic antibiotic therapy (n = 24); and control, no infection and no antibiotic therapy (n = 24). All materials with dimensions of approximately 1 x 1 cm(2) diameter were implanted separately under the dorsal skin of rats. Staphylococcus aureus was used as the infectious agent and antibiotic therapy was done with seftriaxone (Desefin, I.M., 20 mg/kg/day). Tissue specimens were obtained on postoperative days 14 and 21. Semiquantitative and qualitative alterations existing in the connective tissue neighboring the implant material (reaction zone-capsule tissue), fixation to the host tissue, cellular ingrowth (interstice qualitatively), and infection signs were assessed either macroscopically or microscopically. In group A, all materials were affected negatively that led to continuous regression in the wound healing process. Fixation of the cartilage to the surrounding tissue was weak compared with other groups. Fibrovascular tissue ingrowth in porous implants was delayed, and no regular capsule formation was observed around silicone implants. In group B, outcomes were similar to control groups. Porous materials showed tissue ingrowth into the pores as good as the control group. Regular capsular tissue formed around the silicone implants and cartilage tissues. In group C, where silicone had been used, wound healing was not as good as in group B and the control group. In the e-PTFE group, the granulation tissue forming through the pores did not show a good quality as the control group, and capsule formation around the material was irregular, leading to insufficient fixation. While the wound healing properties of the PHDPE group were not as good as the control group, there was no difference in terms of fixation to the wound bed. On the other hand, wound healing of the cartilage group was as satisfying as the control group.

Animals↗
PubMed1988

Radiographic and histologic study of porous coated tibial component fixation in cementless total knee arthroplasty.

Radiographic changes occurring beneath three types of non-cemented, porous coated, cobalt-chrome tibial components were retrospectively reviewed a minimum of 1 year after arthroplasty. Radiolucent zones, radiodense lines, and loose sintered particles were commonly observed. These changes are consistent with non-rigid, fibrous tissue fixation. Histologic evaluation of two specimens retrieved at revision surgery revealed predominantly fibrous tissue within the porous surface under the tibial plateau. The long-term implication of fibrous tissue fixation of porous coated tibial implants in unknown.

Adult↗
PubMed1994

[Optimal conditions for detection of calcitonin mRNA by in situ hybridization (ISH) method using a non-radioactive probe in the rat thyroid gland].

Optimal conditions for detection of calcitonin mRNA were examined by in situ hybridization (ISH) method using a non-radioactive probe in the rat thyroid gland. An oligonucleotide complementary to rat calcitonin mRNA was synthesized with a DNA synthesizer, labelled at 3'-end by using digoxigenin-11-deoxyuridine triphosphate (dUTP) and terminal transferase (Boehringer Mannheim), and used as a probe. Hybrid chains formed by probe and calcitonin mRNA were visualized by anti-digoxigenin-alkaline phosphatase conjugate, nitroblue tetrazolium (NBT) and X-phosphate (Boehringer Mannheim). To determine the optimal conditions for ISH, relations between tissue fixation and proteinase K treatment, and between hybridization temperature and time were mainly surveyed in the present study. As for the relation between tissue fixation and proteinase K treatment, good results were obtained in sections fixed by immersion in 10% formalin at 4 degrees C for 2 hr, and digested with 1 microgram/milliliter proteinase K at 37 degrees C for 20 min. Under this condition, the most intense signals were obtained after hybridization at 37 degrees C overnight. Alternative application of ISH and immunostaining to each of the adjacent sections revealed a small number of cells which were immunonegative but displayed hybridization signals.

Animals↗