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[Metabolism of paraffinic and naphthalenic hydrocarbons in higher animals. I. Retention of paraffins (normal, cyclo and branched) in rats].

Apparent retention of aliphatic hydrocarbons in the Rat is studied. Animals weighing 150 g are put into metabolism cages and given a 15 mg dose of one of the paraffins studied in solution in the peanut oil of the ration. There is no urinary excretion, and fecal elimination is low (5-10%), as long as the carbon chain stays below 24 atoms. Real retention is measured on animals recieving a single dose of 15 mg in the same conditions. Branched paraffins give only very low residual amounts 15 days after ingestion, while n-paraffins and cyclo-paraffins remain unchanged in the carcass, as long as the number of carbon atoms is not greater than 20; the proportion of the latter paraffins is about 8% of the dose ingested (5 days as 21 days after intake). Beyond C 20, real retention decreases rapidly to zero.

Alkanes

Comparison of cytofluorometric nuclear DNA ploidy analysis on fresh and paraffin embedded specimen.

The cytofluorometric nuclear DNA ploidy analysis was performed in gallbladder (GB) epithelium of eight patients of chronic cholecystitis to make a comparison between fresh and paraffin embedded tissue analysis, to make an ideal single cellular suspension, to evaluate the ploidy pattern affected by inflammation and to assess the histological correlations. Six (75%) fresh specimens and eight (100%) paraffin embedded specimens showed diploidy pattern of histogram. Two (25%) fresh specimens showed low ploidy pattern of histogram. There was a good correlation between histological findings and the ploidy patterns of 100% paraffin embedded specimens and 75% fresh specimens. The relative fluorescence intensity was found higher and more stable in paraffin embedded tissues than fresh tissues. Inflammation did not affect the ploidy patterns of paraffin embedded specimens whereas the ploidy patterns of fresh specimens were affected. It can be concluded that the DNA ploidy patterns and histological findings have a good correlation and inflammation does not affect the cytofluorometric DNA ploidy pattern when using the paraffin embedded specimens. Paraffin embedded specimens are superior to fresh specimens for making an ideal single cellular suspension to obtain stable, high, and accurate fluorescence intensity with less background fluorescence which makes the ploidy pattern behave like exact behavior of tissues.

Cholecystitis

A paraffin baiting technique that enables a direct microscopic view of "in situ" morphology of Nocardia asteroides with the acid-fast or fluorescence staining procedures.

The paraffin slide culture technique has provided a means of greatly increasing the effectiveness of the paraffin baiting procedure in the identification of Nocardia asteroides. This has been accomplished with paraffin coated slides rather than the conventional paraffin coated rods, which allows for direct microscopic examination of the "in situ" growth because of its flat surface. This microscopic examination is further facilitated by staining with Kinyoun Acid-fast Stain, or combines three important elements necessary for the identification of Nocardia asteroides, namely; "in situ" morphology, acid-fastness and Auramine fluorescence. The conventional paraffin coated rod only shows the ability of Nocardia asteroides to utilize paraffin as a carbon source, and tells nothing about "in situ" morphology, acid-fastness, and fluorescence with Auramine Fluorochrome.

Bacteriological Techniques

Antidote effect of liquid paraffin in oral solvent intoxication.

The antidote efficacy of liquid paraffin in oral solvent intoxications was investigated in dogs. Groups of 3 to 4 animals each received 1 ml/kg of methylchloroform, 0.3 ml/kg of benzene, or 3 ml/kg of a synthetic gasoline mixture (hexane/heptane/octane, ratio 30:50:20) by gastric intubation. After a prolonged interval the same animals were given 5 ml/kg of liquid paraffin by a separate tube in addition to one of the above solvents. The solvent concentrations in the blood were determined by gas chromatography at appropriate intervals. Administration of liquid paraffin in conjunction with methylchloroform does not significantly affect the absorption process. In the case of benzene and the synthetic gasoline mixture, however, liquid paraffin produces a distinct reduction in the rate of absorption. It is thus concluded that in the therapy of oral solvent-intoxication, liquid paraffin exhibits favorable antidote properties which may however differ from one solvent to the other. In clinically relevant situations involving coadministration of purgatives, the antidote effect must be assessed as being of a higher order than in our animal experiments in which purgatives had to be omitted for external reasons.

Animals

Detection of hepatitis C viral RNA sequences in fresh and paraffin-embedded liver biopsy specimens of non-A, non-B hepatitis patients.

In this study methods of HCV-RNA detection in fresh frozen and formalin-fixed, paraffin-embedded liver biopsies are described. Of 22 untreated chronic non-A, non-B hepatitis patients and 6 control patients, a plasma sample and part of a liver biopsy were freshly frozen for hepatitis C virus (HCV) cDNA-PCR. From 16 of the same non-A, non-B hepatitis patients and from 5 of the same control patients formalin-fixed, paraffin-embedded liver tissue from the same biopsy was available also for HCV cDNA-PCR. In 13 of 22 non-A, non-B hepatitis patients HCV-RNA could be detected in plasma as well as in liver tissue. In the other 9 non-A, non-B hepatitis patients and in 6 control patients, no HCV-RNA was detectable in either plasma or liver tissue. The comparison between HCV cDNA-PCR results in fresh frozen versus formalin-fixed, paraffin-embedded liver biopsies showed that although detection of HCV-RNA in both correlated 100% the quantity of HCV-RNA was lower in the formalin-fixed, paraffin-embedded liver biopsies of 5 of 8 patients for whom end-point dilution titration of liver RNA was performed. We conclude that using the procedures described HCV-RNA can be reliably detected in both fresh-frozen and formalin-fixed, paraffin-embedded liver biopsies and that HCV cDNA-PCR in liver tissue may become an important assay, especially for monitoring anti-viral therapy.

Biopsy

Demonstration of immunoglobulin in cryostat and paraffin sections of human tonsil by immunofluorescence and immunoperoxidase techniques. Effects of processing on immunohistochemical performance of tissues and on the use of proteolytic enzymes to unmask antigens in sections.

A fluorescein isothiocyanate (FITC) technique and one based on peroxidase-antiperoxidase (PAP) were used to study the distribution of immunoglobulin (Ig) in cryostat and paraffin sections of human tonsil. Trypsin and other proteolytic enzymes were used to 'unmask' the antigen in paraffin sections. The effects of processing, and particularly of fixation, on the immunohistochemical response of tissues were studied. The FITC and PAP methods detected Ig in paraffin and cryostat sections equally well. The distribution of the antigen was the same with both methods but the PAP method was the more informative. Formaldehyde-sucrose solution proved more suitable for fixing tissues for immunohistochemistry than glutaraldehyde. Trypsin revealed antigen in parraffin sections more efficiently than pepsin, papain, or pronase. Surface Ig (s-Ig) could be demonstrated in trypsinised paraffin sections but less effectively than in cryostat sections. Trypsinised paraffin sections were, however, more suitable for intracellular Ig (c-Ig) than cryostat sections although the performance of cryostat sections could be improved by prior fixation with a coagulative fixative.

Antigens

Molecular pathology of paraffin-embedded tissue. Current clinical applications.

Molecular biology techniques have been adapted to analyze paraffin-embedded tissues, expanding their potential clinical utility. The isolation of intact nucleic acids from tissue blocks is fundamental to the molecular pathology of paraffin-embedded tissues. In vitro amplification with the polymerase chain reaction (PCR) promises to be the most useful means of retrospective analysis since it can be performed successfully on DNA that has partially degraded during fixation, paraffin embedding, and the extraction process. Four clinical situations in which DNA analysis of paraffin-embedded tissues can be helpful are: (a) gene rearrangement analysis in lymphoproliferative disorders, where fresh tissue has not been obtained at the time of surgery; (b) identification of infectious agents, particularly viruses; (c) genetic testing of families with a putative inherited disease where the affected member has died; and (d) specimen identification. The PCR and other techniques of genetic analysis are powerful in sensitivity and specificity when performed and interpreted with appropriate precautions and controls. DNA analysis of paraffin-embedded tissues will likely become a fixed part of the future pathologist's diagnostic armamentarium.

DNA

Paraffin oil pneumonia. Analysis of saturated hydrocarbons in different human tissue.

Temperature-programmed gas chromatographic analysis on columns packed with Apiezon L as stationary phase is shown to be the best method for the qualitative and quantitative analysis of simple and complex hydrocarbon mixtures when compared with all the other applicable techniques (thin-layer chromatography, column chromatography, ultraviolet spectroscopy, infrared spectroscopy, nuclear magnetic resonance spectroscopy, mass spectrometry) described in this paper. Using the method in a patient with mineral oil pneumonia it could be demonstrated that he expectorated a maximum of 79.5 mg liquid paraffin daily and also transported equally complex saturated hydrocarbons in a concentration of 1.3 mg% in plasma and of 1.6 mg% in the cellular blood components. In an additional experiment the direct determination of liquid paraffin resorbed from the gastrointestinal tract was possible in a patient with a left chyle fistula in the neck. After a dose of 50 g liquid paraffin administered as a laxative, 246 ml chyle was collected within the following 14 h which yielded a total of 4.5 mg liquid paraffin. Its composition was identical with the administered laxative. Assuming a daily lymph volume of 1.51, the resorbed amount would correspond to a resorption rate of 0.5 (see article) liquid paraffin. The importance of these results as well as the diagnostic consequences arising from the described analytical technique are discussed in detail.

Adult

Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections.

The monoclonal antibody Ki-67 reacts with a human nuclear cell proliferation-associated antigen that is expressed in all active parts of the cell cycle. Recently we have raised monoclonal antibodies, MIB 1-3, against recombinant parts of the Ki-67 antigen. These antibodies are true Ki-67 equivalents, as demonstrated by immunostaining of fresh specimens, biochemistry, and molecular biological techniques. Formalin-fixed, paraffin-embedded sections routinely processed for immunohistochemistry failed to stain for Ki-67 and MIB 2. Antibodies MIB 1 and MIB 3 labelled mitotic figures, while non-mitotic proliferating cells were negative under these conditions. However, when dewaxed microwave oven-processed paraffin sections of formalin-fixed tissues were used, MIB 1 and MIB 3 gave strong nuclear staining of those cells presumed to proliferate under a variety of normal and neoplastic conditions. Moreover, routine decalcification or depigmentation techniques did not alter the immunoreactivity of MIB 1 and MIB 3 with microwave-processed paraffin sections. This method is highly reproducible, easy to perform at low cost, and no additional technical skill is needed because after microwave treatment just routine immunohistochemical methods are used. Since we have successfully applied this new method to sections obtained from paraffin blocks stored for a long time (in one case more than 60 years), the assessment of cell kinetics through the detection of Ki-67 antigen is now possible on archival material collected in histopathology departments all over the world.

Antibodies, Monoclonal

Cryofixed, freeze-dried and paraffin-embedded skin enables successful immunohistochemical staining of skin basement membrane antigens.

Conventional chemical fixation and paraffin-embedding procedures give good preservation of morphology, although the antigenicity of many proteins in the tissue sample is destroyed. On the other hand, fresh frozen sections can preserve the antigenicity, but provide poor morphological preservation. To overcome this dilemma, cryofixation and freeze drying were used on human skin tissue, applying methodology which has only been used to study lymphoid tissue. First, fresh human skin was cryofixed in liquid isopentane (-160 degrees C) cooled by liquid nitrogen. The skin was then freeze-dried at -40 degrees C and 10(-2) atmospheric pressure for 72 h, followed by embedding in paraffin. Sections 4 microns thick taken from this cryofixed, freeze-dried, and paraffin-embedded skin were stained with hematoxylin-eosin or used for immunolabeling with antibodies against basement membrane antigen, including type IV and type VII collagen, bullous pemphigoid antigen, epidermolysis bullosa acquisita antigen, and GB3 antigen. The morphological preservation of these sections was as good as that of routine formalin-fixed and paraffin-embedded skin sections. The basement membrane was clearly immunostained with all antibodies used, and the intensity of the reaction was as strong as that seen in frozen sections. Evaluation of antigen distribution in conjunction with the detailed skin structure was therefore possible in the same sections.

Antibodies, Monoclonal

Immunofluorescent detection of hepatitis B antigen in paraffin-embedded liver tissue.

Hepatitis B antigen (HBAg) has been demonstrated by the indirect immunofluorescent technique and by orcein staining in 20 liver biopsies fixed in Bouin's fixative and embedded in paraffin. The results were compared with those obtained previously by immunogluorescence on frozen sections of the same biopsies. Ten biopsies which were positive in frozen sections were also positive by immunofluorescence in parafin sections, whereas only six were positive by orcein staining. In orcein-stained sections, the cellular localization of HBAg was precisely in the same places as in the slides examined by immunogluorescence. The intessity of the fluorescence in paraffin sections was almost the same as in frozen sections. The localization of the antigen was histologically more precise in paraffin sections. Besides various advantages, indlucing aboidance of freezing aquipment and procedures, paraffin sections are more easy to handle and biopsies from distant hospitals can be processed. The advantages of the immunofluorescent test in comparison to orcein staining are its immunological specificity and higher sensitivity.

Biopsy

Anti-CD34 immunoperoxidase staining in paraffin sections of acute leukemia: comparison with flow cytometric immunophenotyping.

Anti-CD34 is a monoclonal antibody that reacts with bone marrow progenitor cells and leukemic blasts, and is expressed on 30% to 50% of all acute leukemias. Detection of CD34 has previously been restricted to flow cytometric studies. To expand the utility of CD34, we immunostained 46 paraffin-embedded bone marrow specimens with acute leukemia; results were compared with flow cytometric studies. CD34 reactivity was also evaluated in nine chronic leukemia cases, 27 malignant lymphoma cases (Hodgkin's disease and non-Hodgkin's lymphoma), six normal bone marrow specimens, and three benign, hyperplastic lymph node specimens. All cases that were CD34 positive by flow cytometry (11 of 19 B-cell precursor acute lymphoblastic leukemia cases, one of six T-cell acute lymphoblastic leukemia cases, and seven of 21 acute myeloblastic leukemia cases) were also CD34 positive in paraffin sections. Both cell membrane and cytoplasmic staining was seen. The positivity percentage and fluorescence intensity by flow cytometry correlated with the estimated number of stained cells and the intensity of immunoperoxidase staining in 18 of 19 CD34-positive cases. The remaining bone marrow and lymph node cases studied were CD34 negative; prominent endothelial cell staining, however, was noted. This is the first report of anti-CD34 staining of acute leukemia in paraffin-embedded sections. In contrast to other monoclonal antibodies reactive in bone marrow paraffin sections with leukemia, anti-CD34 immunoperoxidase staining is limited to leukemic blasts and may provide useful diagnostic information when flow cytometric studies are not available.

Acute Disease

A new metachromatic stain technique for paraffin-embedded neural tissue using thionin.

A metachromatic staining procedure which differentiates Nissl substance in neuron cell bodies, neuropil area, and axonic fibres of passage with distinct colours with no counterstaining has been developed for paraffin-embedded tissue utilizing thionin stain. Frozen sections of neural tissue fixed in formalin may also be satisfactorily stained by this procedure, and both paraffin-embedded and frozen sections have been found to retain the distinct colours for a year with little fading. Since metachromatic staining of paraffin-embedded neural tissue has not been successfully achieved before, the following procedure will be especially valuable for studying small vertebrate brains, or other central nervous tissue which must be processed by the paraffin method.

Animals

Immunofluorescent detection of alpha1-antitrypsin in paraffin embedded liver tissue.

Alpha1-antitrypsin was detected by indirect immunofluorescence in frozen sections of liver biopsies from patients with clinically and biochemically proven alpha1-antitrypsin deficiency. The antigen could also be demonstrated in those liver specimens of the same patients which were fixed in Bouin's fluid and embedded in paraffin. The cellular localization and the brightness of the fluorescence were the same in both frozen and paraffin sections. Four additional biopsies from three other patients were selected on the basis of PAS-positive diastase-resistant inclusions reported in the hepatocytes. All these biopsies showed bright fluorescence in the cytoplasm of the liver cells although one of the biopsies was stored for as long as eight years. Specific fluorescence was constantly found in the periportal hepatocytes with varying degrees of positivity. No fluorescence was observed in the six control biopsies from patients with various other liver diseases. These findings prove that paraffin embedded specimens are suitable for immunofluorescence detection of alpha1-antitrypsin and that a retrospective study on old paraffin blocks is possible.

Adult

Immunohistochemical detection of estrogen receptors on paraffin sections of normal, hyperplatic and carcinomatous endometrium.

The present study is the first dealing with the demonstration of estrogen receptors (ER) in up to 8-year-old paraffin blocks of endometrial curettage samples routinely fixed in 10% formalin. The Mab ER-ICA was used in a modified peroxidase-antiperoxidase method after pretreatment of paraffin sections with pronase. Eleven cases with proliferative, 11 cases with secretory endometrium, 20 cases with adenocystic, 21 with adenomatous hyperplasia and 27 endometrial adenocarcinomas were tested. The two main parameters, namely the percentage of ER-positive cells and the intensity of the immunostaining, were higher in the proliferative phase followed in a declining sequence by adenocystic hyperplasia, adenomatous hyperplasia, adenocarcinomas and the secretory phase of endometrium. Interestingly, the intensity of the immunostaining showed a positive relationship to the percentage of ER-positive cells (r = 0.93, p less than 0.001). It seems that the immunohistochemical demonstration of ER in paraffin sections of uterine specimens is an easy and reliable method for the mapping of the heterogeneous expression of ER and their comparative study with the well preserved histopathological features even in old archival paraffin-embedded material.

Adenocarcinoma