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PubMed1995

[Techniques for the diagnosis of kidney diseases. Is there a role for the immunoperoxidase method in the evaluation of immunologic glomerulopathy on biopsy specimens, including those in paraffin? Our experience: preliminary results].

Firstly an extensive summary of the diagnostic power and limits in renal glomerular pathology of light microscopy even corroborated by all the special histochemical stains is made. Secondly a complete list of all those renal glomerular diseases in which electron microscopy is unanimously considered necessary for the diagnosis is given as well as the main and most updated references pertinent to those rare and recently recognized pathological entities in which EM proved as a fundamental diagnostic tool are provided. Thirdly the authors focus on the importance of immunohistochemistry (IIC) in the diagnosis of immune-mediated renal glomerular diseases. The advantages and disadvantages of immunofluorescence microscopy (IF) and immunoperoxidase method (I-perox) are respectively discussed. The value of I-perox staining method applied to sections from paraffin blocks is emphasized and some technical suggestions in order to optimize results are given. Among the latter the choice of the used fixative for the purpose of preserving antigenicity of immunoglobulins and complement fractions, the timing of tissue fixation as well as the quantitation of enzymatic pretreatment relative to the duration of fixation, and the repeated washings with phosphate-buffer saline to minimize background staining. Ultimately the authors report on their preliminary personal results with the use of I-perox staining on sections from paraffin embedded renal core biopsies. Although if is their favourite diagnostic microscopy for evaluating renal immunopathology the authors underline I-perox method as a helpful tool to rely on at least in those circumstances when frozen material for IF is not available due to the scarcity of core biopsy, when glomeruli are absent in frozen sections, or when due to a technical lab accident the frozen tissue devoted to IF has been ruined or lost, and the only available material is the one which lies embedded in paraffin. The LSAB ("labelled streptavidin biotin") complex and PAP ("peroxidase-antiperoxidase") working procedures are provided in detail.

Biomarkers↗
PubMed1987

Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities.

Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneously demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3-7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH 7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗
PubMed1995

Microwave-enhanced in situ end-labeling of fragmented DNA: parametric studies in relation to postmortem delay and fixation of rat and human brain.

In situ end-labeling (ISEL) identifies DNA fragmentation in apoptotic or necrotic nuclei in tissue sections. However, application of ISEL on human brain requires conservation of DNA integrity during the postmortem delay (PMD) and good accessibility of fragmented DNA after (prolonged) tissue fixation. We therefore investigated ISEL in relation to PMD and fixation in rat and human brain. Application on a unilateral lesion model in perfused rat brain revealed that prolonged post-fixation strongly diminished ISEL results. However, microwave pre-treatment can counteract these masking effects without inducing nonspecific labeling contralaterally. On the other hand, in briefly post-fixed, perfused brain or immersion-fixed rat and human PMD brain, microwave pre-treatment was deleterious and induced strong nonspecific labeling. In young rat brain, PMD did not influence the low numbers of apoptotic nuclei until 24 hr PMD, when massive nuclear labeling occurred. In human cortex, DNA fragmentation patterns were independent of duration of fixation or PMD and were already present from 4.25 hr PMD onwards. Our data suggest that ISEL on human brain represents antemortem DNA damage rather than PMD artifacts. Furthermore, microwave pre-treatment appears beneficial only in particular fixation conditions.

Aged↗
PubMed1995

ImmunoMax. A maximized immunohistochemical method for the retrieval and enhancement of hidden antigens.

BACKGROUND: Since the introduction of mAb, immunohistochemistry has become an important tool in research and in surgical pathology. The most widely used fixative in routine histopathology is formaldehyde, and it has become the gold standard for morphologic tissue preservation. Although the molecular mechanism underlying the tissue fixation is not well understood, it has become clear that available immunoreactive Ag are progressively lost during the fixation process. For a long time, it was thought that formalin-sensitive Ag might be irreversibly destroyed during the fixation process. Although monoclonal anti-Ig Ab frequently worked inadequately, polyclonal anti-Ig Ab were shown to produce reproducible staining results. It thus appeared possible that most cellular Ag might not be irreversibly destroyed but only masked. EXPERIMENTAL DESIGN: Although some Ag may be retrieved under appropriate conditions, there might still be many for which available antigenic epitopes are still too sparse to be visualized, as observed for a large number of leukocyte differentiation Ag. One reliable approach to resolve this dilemma is the use of a combination of an optimized Ag retrieval system and a powerful immunohistochemical staining protocol introducing a biotin amplification step, in which signal amplification is accomplished by covalent deposition of biotin molecules. RESULTS: Cryostat and paraffin sections were stained with the avidin-biotin complex technique and, for comparison, with the new maximized immunohistochemical staining protocol, termed the ImmunoMax method. Each step was monitored to establish how effectively it enhanced the overall sensitivity. Although pretreatment with detergent, protease, a chaotropic substance, or microwave heating resulted in only moderately improved immunostaining, the biotinylated tyramine enhancement step proved to be the most efficient one, although the latter is not sufficient for many Ag when used without pretreatment steps. The combination of an Ag retrieval step with the biotinylated tyramine enhancement step resulted in a 100 to 10,000-fold boost in sensitivity without loss of specificity. CONCLUSIONS: With the ImmunoMax method, defined Ag can be reproducibly detected in formalin-fixed, paraffin-embedded tissues, and the sensitivity of the method is tremendously enhanced. Moreover, it also allows many previously unreactive or unsatisfactorily reactive Ag to be detected, as shown here for IgD, IgM, and CD7 with the use of mAb.

Antigens↗
PubMed2005

Reverse achilles tendon allograft technique for anterior cruciate ligament reconstruction.

Because of the increasing popularity of allograft anterior cruciate ligament (ACL) reconstructions, the technical difficulties with posterior bone loss in revision ACL surgery, and the limited supply of bone-patellar tendon-bone donor grafts, we have developed a technique using a reversed Achilles tendon allograft to reconstruct the ACL-deficient knee. This technique allows for bony tibial fixation with an interference screw by rotating the graft 180 degrees and optimizing soft-tissue fixation at the femur with an EndoButton CL (Smith & Nephew, Andover, MA). Short-term follow-up (average, 26.4 weeks) of 10 patients undergoing the reversed Achilles technique has shown excellent results, with Lysholm scores between 91 and 100 and 84 and 90 in 8 of 10 and 2 of 10 patients, respectively. Objective stability assessments, including the Lachman test, anterior drawer, and pivot-shift tests, were also encouraging. Reverse Achilles tendon allografts can be excellent alternatives for ACL reconstruction when bone-patellar tendon-bone grafts are not indicated.

Achilles Tendon↗
PubMed1984

A method for exposing the internal anatomy of small and delicate tissues for correlated SEM/TEM studies using polyethylene glycol embedding.

A method for preparing and handling large, clean, distortion-free cut surfaces through small and delicate tissues for correlated SEM/TEM examination is described. In this method, tissues are fixed according to conventional protocols; however, instead of critical-point-drying after fixation, tissues are first embedded in polyethylene glycol (PEG), a water-soluble waxy solid. Tissue blocks are easily oriented and sectioned to the desired regions, immersed in a solvent to remove PEG, critical-point-dried, and examined with an SEM. The same tissue blocks can be reworked for TEM by immersing in propylene oxide and embedding in an epoxy resin.

Animals↗
PubMed1995

Stathmin expression is a feature of proliferating cells of most, if not all, cell lineages.

BACKGROUND: Stathmin is a phylogenetically conserved protein which was identified initially as a prominent cytosolic protein in hemopoietic cells, endocrine cells, brain, and testis. In these tissues, it has been suggested that the level of stathmin expression is important in development and cell proliferation. Furthermore, stathmin phosphorylation appears to be involved in the regulation of cell growth arrest, terminal differentiation, and hormone secretion. Elevated levels of expression of stathmin have been described in leukemia and lymphoma cells. The aim of this study was to characterize the distribution of cells that express the stathmin protein in a wide variety of normal human and rodent tissues. EXPERIMENTAL DESIGN: First, antisera against a synthetic stathmin peptide have been raised in rabbits and the specificity of these antisera confirmed by their reactivity with stathmin on 1-dimensional and 2-dimensional Western blots. Second, the most appropriate means of fixing tissues in order to stain stathmin has been investigated. Finally, using the optimized conditions of tissue fixation, the antisera have been used to immunostain sections taken from a wide variety of tissues. RESULTS: Immunopositivity was found in cells of all the lineages studied, with the stained cells present within the proliferating compartment of tissues. Conversely, most nonproliferating mature cells did not stain with the antisera to stathmin. The only nonproliferating cells that appeared to express stathmin were a subpopulation of glial cells, neurons, and anterior pituitary cells. CONCLUSIONS: It is proposed that stathmin is necessary for cell proliferation in most, or all, cell lineages and that its primary function relates to some aspect of cell division.

Animals↗
PubMed1991

Histochemical demonstration of sinusoidal gamma-glutamyltransferase activity by substrate protection fixation: comparative studies in rat and guinea pig liver.

Most histochemical methods for the detection of an enzymatic activity are preceded by tissue fixation with chemical agents that partially inactivate the enzymes. It is well known that substrates exert a marked protection against fixative-induced inactivation. The conventional histochemical methods for the demonstration of hepatic gamma-glutamyltransferase activity have not been successful in detecting the activity of the enzyme on the sinusoidal side of the hepatocytes despite mounting biochemical evidence for its presence on that pole of the hepatocyte. Under conventional fixation the enzymatic activity in hepatocytes is only seen on the bile canalicular side. This may be the result of a preferential protective effect of gamma-glutamyltransferase by its normal substrate, glutathione, present in the bile canaliculus at concentrations 500 times higher than in the sinusoidal lumen (8 mmol/L vs. 10 to 20 mumol/L). To test this hypothesis and to reduce the degree of fixative-induced inhibition of the enzyme activity, glutathione was either incorporated in the fixative solution or the livers were perfused with high concentrations of glutathione (10 mmol/L) before fixation. Our results histochemically demonstrate, in the normal adult rat liver, the existence of gamma-glutamyltransferase activity not only on the bile canalicular pole but also on the sinusoidal pole of the hepatocytes. Visualization of the enzyme activity on the sinusoidal pole is dependent on glutathione protection. Guinea pig livers, which present a 10-fold higher gamma-glutamyltransferase activity than rat livers (similar to that in human beings), showed marked sinusoidal gamma-glutamyltransferase activity even in the absence of glutathione protection. Glutathione protection further increased this sinusoidal activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

[Detection of fibronectin and collagen types I, III, IV and V in semithin sections of the human aorta].

Localization of fibronectin and types I, III, IV and V collagen was investigated in semithin sections of fibrous atherosclerotic plaques and apparently normal intima of human aorta. The effect of different techniques of fixation and processing of the sections on immunostaining under peroxidase-antiperoxidase techniques have been examined. The tissue fixation in paraformaldehyde solution, removing the resin with sodium ethoxide and enzymatic pronase digestion of the sections resulted in successful specific staining of the antigens. It was found that some cells in fibrous plaques formed a cap of multiple layers of dense connective tissue containing fibronectin and type III, IV and V collagen in the absence of collagen type I.

Adult↗
PubMed1996

Comparison of the UniversalKeratome and the Automated Corneal Shaper.

PURPOSE: To compare the morphologic appearance and measurements of in situ keratomileusis performed with the UniversalKeratome (UK) with those done with the Automated Corneal Shaper (ACS). SETTING: Surgical suite within private practice. METHODS: Procedures were performed the same day on mate eye-bank eyes. In situ keratomileusis was done using existing nomograms for each instrument to resect a cap thickness of 160 microns and a myopic resection of 100 microns. Intraocular pressures were increased by inflating the globes with balanced salt solution and were measured with the suction fixation rings in place. The excised caps and stromal resections were measured twice independently after surgery, again after tissue fixation, and then evaluated with light and scanning electron microscopy. RESULTS: No complications were encountered. Compared with the ACS, the UK was easy to set up, use, clean, and take down. Its excised tissue dimensions were greater and more predictable, it resected a concave shaped lenticule (edges imperceptibly blending with the host stroma), and it created a smoother power resection surface and primary resection base. CONCLUSIONS: Smoother, predictable tissue resection, and simple assembly/disassembly and use give the UK an apparent advantage over the ACS. The UK corrects astigmatism and hyperopia by changing the shape of the poly(methyl methacrylate) optical insert.

Aged↗
PubMed1983

Histochemical localization of neuraminidase in the CNS of mice and fish by means of 5-brom-3-indolyl-alpha-ketoside of 5-N-acetyl-D-neuraminic acid (BI-NeuAc).

The applicability of the 5-brom-3-indolyl-alpha-ketoside of N-acetyl-D-neuraminic acid (BI-NeuAc) as substrate for the histochemical indication of neuraminidase (GOSSRAU et al. 1977) was examined in frozen sections of brain, small intestine and kidney from suckling and adult mice and respectively from a cichlid fish (Sarotherodon mosambicus) with and without formaldehyde fixation. Following biochemical investigations using tritiated gangliosides as substrate a decrease in the activity of neuraminidase took place from about 50 to 80% after tissue fixation with formaldehyde. Nevertheless significant histochemical reactions were found only in fixed tissue sections. The specificity of this reaction was shown by means of inhibitions tests using 2,3-dehydro-2-desoxy-N-acetyl-neuraminic acid according to KUMAR et al. (1981). In contrast to distinct staining of neuraminidase activity in the small intestine and kidney, only faint reactions occurred in brain sections. From this it is concluded that the application of the new synthetic substrate for quantitative staining of neuraminidase seems to be not very suitable for the CNS.

Animals↗
PubMed1991

Basement membrane proteins in the space of Disse: a reappraisal.

The distribution of two major basement membrane components, type IV collagen and laminin, was studied within the perisinusoidal space of Disse in normal human liver using (i) an immunoperoxidase method for light microscopy and (ii) immunogold labelling for ultrastructural localisation. Although immunoreactivity depended on the mode of tissue fixation, both proteins could be identified at this site using a panel of affinity purified antibodies. These findings indicate that these proteins are normal constituents of the perisinusoidal extracellular matrix, and refute the hypothesis that capillarization of the sinusoids in chronic liver disease results from neo-expression of laminin in the space of Disse.

Basement Membrane↗
PubMed1990

[Ultrastructural localization of myelin bodies and acid phosphatase activities in the liver and kidney induced by quinacrine in rats].

Electron microscopic studies were conducted to reveal the ultrastructural aspects of the myelin body and acid phosphatase activity in rats induced by quinacrine, an antimalarial drug. Each of 22 rats in three groups were examined. The first group of control rats was initially given a single i.p. dose (200 mg/kg) of diethylnitrosamine (DEN) only and then fed a CRF-1 basal diet for 8 weeks. The second and third group were treated with DEN or saline, and starting 2 weeks later, were fed a CRF-1 basal diet supplemented with 500 ppm quinacrine for 6 weeks. All animals were subjected to a partial hepatectomy at week 3, and then sacrificed at week 8. Liver and kidney tissues specimens from 2 rats per group were collected for routine electron microscopic study. Furthermore, the activity of acid phosphatase, a key enzyme for lysosomal activity, was also investigated. In this study, tissues were fixed with a solution of 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer. After fixation, tissues were frozen and their 8 microns sections were treated with Gomori's lead nitrate buffer solution, and post-fixed with a 1% osmium solution. After being embedded in Epon 812, ultrathin sections were made. Intracellular myelin bodies were observed in the hepatocytes, interlobular bile duct cells, renal glomerular podocytes and renal tubular cells in the quinacrine treated groups, but not were observed in rats treated with DEN alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗
PubMed1987

Porous-coated hip replacement. The factors governing bone ingrowth, stress shielding, and clinical results.

Total hip replacement using porous-coated cobalt-chrome femoral implants designed for biological fixation has been evaluated in 307 patients after two years and in 89 patients after five years. Histological study of 11 retrieved specimens showed bone ingrowth in nine and fibrous tissue fixation in two. Fixation by bone ingrowth occurred in 93% of the cases in which a press fit of the stem at the isthmus was achieved, but in only 69% of those without a press fit. The clinical results at two years were excellent. The incidence of pain and limp was much lower when there was either a press fit of the stem or radiographic evidence of bone ingrowth. Factors such as age, sex, and the disease process did not influence the clinical results. Most cases showed only slight resorptive remodelling of the upper femur, but in a few cases with a larger, more rigid stem, more extensive bone loss occurred. The results after five years showed no deterioration with time. Fixation by the ingrowth of bone or of fibrous tissue both appeared to be stable, but bone ingrowth gave better clinical results.

Aged↗
PubMed1988

Differential sensitivity of the microtubule-associated protein, tau, in Alzheimer's disease tissue to formalin fixation.

Immunohistochemistry of formalin-fixed human Alzheimer's disease (AD) tissue using an anti-tau antibody (Tau-1) reveals staining of neurofibrillary tangles (NFTs) and neuritic plaques (NPs), whereas normal axonal staining is less apparent. In this study, we used a combined biochemical and histochemical approach to assess effects of formalin on immunoreactivity of AD tau. Nitrocellulose blots were treated with fixative to mimic conditions used with tissue sections, a method that might be generally useful for assessing antigen sensitivity to different fixatives. A progressive decrease in Tau-1 immunoreactivity of the tau bands on a Western blot was observed with increasing times of formalin fixation. Phosphatase-digested blots demonstrated an increase in Tau-1 immunoreactivity compared to control blots. These results mimic the phosphatase-sensitive Tau-1 immunohistochemical staining of formalin-fixed AD tissue slices previously reported. Fixation of AD tissue with periodate-lysine-paraformaldehyde (PLP) preserves axonal tau antigenicity. Phosphatase digestion of PLP-fixed AD tissue enhances Tau-1 immunoreactivity of NFTs and NPs but does not alter axonal staining. These results indicate that axonal form(s) of tau are more sensitive to formalin fixation than pathology-associated tau. In addition, a modification of AD tau in pathological structures may protect it from the effects of formalin with regard to Tau-1 antigenicity.

Alzheimer Disease↗
PubMed1980

Detection of human chorionic gonadotropin in fresh and formalin-fixed testicular tumor tissue. Comparison of sensitivity of immunoperoxidase to radioimmunoassay.

Immunmocytochemical techniques for the detection of human chorionic gonadotropin (HCG), such as immunoperoxidase or immunofluorescence, allow detection but not quantification of this hormone. A modification of standard radioimmunoassay (RIA) techniques allows utilization of tissue slurries and yields quantitative data on tissue HCG levels. Tissue slurry RIAs and indirect immunoperoxidase were performed on fresh and formalin-fixed tissue specimens from 15 patients with nonseminomatous testicular tumors. HCG detectability by RIA or immunoperoxidase was markedly decreased by formalin fixation. Tissue specimens with high levels of HCG in the fresh state yielded positive immunoperoxidase and RIA results, while specimens with minimally elevated HCG levels tended to be negative. Results of tissue slurry RIAs correlated well with immunoperoxidase results. The detection of HCG in fixed tissue appears to underestimate the amount of HCG present in fresh specimens. Therefore, a negative RIA on formalin-fixed tissue slurry or negative immunoperoxidase of the formalin-fixed, paraffin-embedded tissue does not rule out the absence of HCG in fresh tissue.

Chorionic Gonadotropin↗
PubMed2004

The role of soft tissues in plate fixation of proximal phalanx fractures.

The tension band effect of plate fixation and the contribution of soft tissues to that effect was examined biomechanically in human proximal phalanges. Forty-six proximal phalanges in whole cadaver hands with all soft tissues in place (intact) and 43 proximal phalanges stripped of soft tissues (denuded) were tested. After midshaft osteotomy, each proximal phalanx was fixed internally with a dorsal minicondylar plate, a lateral minicondylar plate, a dorsal straight plate, or a lateral straight plate. Specimens were tested in three-point apex dorsal bending to clinical failure, defined as 30 degrees angulation. Ultimate moment (stability) at this angulation was similar among the four fixation methods in the specimens with all soft tissues intact. Stability also was similar among these methods in the denuded specimens. There were no significant differences in stability between minicondylar and straight plates or between dorsal and lateral plates in the specimens with soft tissues, nor were there significant differences between these groups in the denuded specimens. The stability of the four fixation methods was significantly greater in the specimens with soft tissues than in the denuded specimens. Soft tissues increased the stability of lateral minicondylar plates by 163%, lateral straight plates by 157%, dorsal minicondylar plates by 126%, and dorsal straight plates by 104%, providing a dorsal tension band effect that counteracted the buttress (compression) of the volar fracture surfaces of the phalanx. The results suggest that in the clinical setting a laterally placed straight or minicondylar plate may provide as much stability to a phalanx with a midshaft fracture as does the traditional, more invasive dorsally placed minicondylar or straight plate. These findings must be evaluated with caution, however, because all specimens were from embalmed cadavers, and the formalin fixation may have augmented the stability and stiffness of the soft tissues in the intact specimens. A subsequent pilot study comparing intact proximal phalangeal specimens that were formalin-fixed with those that were fresh-frozen showed a significant increase in stability and stiffness of formalin-fixed specimens.

Biomechanical Phenomena↗
PubMed1990

A simple and effective method for inactivating virus infectivity in formalin-fixed tissue samples from patients with Creutzfeldt-Jakob disease.

We fixed brains from hamsters infected with scrapie virus in (1) formalin, (2) phenol-saturated formalin, (3) formalin with a 1-hour immersion in formic acid, or (4) phenol-saturated formalin with a 1-hour immersion in formic acid. In addition, we used the formalin-formic acid procedure on brains from mice infected with the virus of Creutzfeldt-Jakob disease. Formic acid proved superior to phenol in respect to both disinfection and tissue preservation, almost completely eliminating virus infectivity in sections that were histologically indistinguishable from formalin-fixed material. The inclusion of a formic acid step in routine formaldehyde tissue fixation will thus provide histologic sections of excellent quality, and virtually eliminate the risk of handling infectious material in the subsequent neuropathologic processing of tissues from patients with CJD.

Animals↗