Double Embedding Technique Research Articles

Conventional paraffin embedding technique and double-embedding technique for minute oral biopsies and delicate pulp tissue - a comparative study

Among oral biopsies, small incisional tissues, have to be preserved all through the processing and embedding to ensure optimal visualization of all the mucosal layers without compromise. Optimal tissue orientation is the most critical step in tissue processing for demonstration of definitive morphology in the sections, which is often more challenging in cases of minute/small or thinner sections using routine paraffin techniques to evaluate accurate diagnosis. Some modification is needed to handle these samples to get a better result. Double embedding technique with some modification has been widely used for small/ thin/ multiple biopsies and gives excellent results in many other fields like general pathology and biotechnology. The double embedding technique though produced excellent and significant results in mucosal biopsies yet, it is of minimal interest among oral pathologists. To best of our knowledge, this is the first study to use double embedding technique for pulp tissues. The present study was aimed to evaluate and compare the ease of embedding and sectioning sections using Agar-Paraffin double embedding technique for small oral mucosal biopsies and thin pulp tissues. A total of 40 oral tissue samples categorized into two groups were taken for the present study. Group I included 20 small oral mucosal biopsy samples of size ranging from 0.2 to 0.5 cm and Group II included 20 pulp tissues obtained from freshly extracted non carious tooth. 10 blocks were prepared by routine paraffin method and 10 blocks were prepared by modified double embedding method for each group. Scores were given by comparing all the criteria with that of the routine paraffin technique. Chi-square test was used for statistical analysis. The average ease score for the Agar-Paraffin double embedded small/minute biopsies showed better scores than the pulp tissue with that of the routine technique. However, no statistically significant difference was seen among embedding and sectioning sections between the two groups. Modified double embedding method is simple and reliable alternative technique that helps in better orientation, processing and sectioning especially for oral small or thin biopsies and delicate pulp tissues.

An Approach for Aspect Extraction Using Double Embedding Technique based Machine Learning Model

An Approach for Aspect Extraction Using Double Embedding Technique based Machine Learning Model

Agar-Paraffin Double Embedding Over Conventional Embedding for Minute Oral Biopsies- Cohort Study

Agar-Paraffin double embedding technique is a simple technique that combines the advantages of both the embedding material. It preserves the minute tissue biopsies in orientation and holds them together from getting lost. Oral biopsies, being most commonly small incisional tissues, have to be preserved all through the processing and embedding to ensure optimal visualization of all the mucosal layers without compromise. In the present study, samples were divided into two groups: Group A: Agar-paraffin double embedding (APE) and Group B: conventional paraffin embedding (PE). Tissue samples collected were sectioned into two approximately equal sized bits. Both the bits were simultaneously processed, embedded in two different techniques (APE and PE method). Sections obtained were scored by an observer and analyzed using independent sample t-test (SPSS software version 21) to evaluate the efficacy of agar-paraffin double embedding technique in comparison to the conventional paraffin embedding technique. Agar paraffin embedded tissue was found to be well processed, firm and well preserved. Orientation was comparatively easier and the blocks yielded sections of good quality. They showed no interference with staining and cell morphology was of good clarity. Thus Agar-paraffin embedding technique represents a simple, reliable method that can greatly improve the quality of diagnostic information.

Surgical Inking and Double Embedding Technique for Routine Histopathology to Save Time and Effort

Background: Routine histopathological specimens like appendix and fallopian tube require individual processing, block making and slide preparation. This process is not only time consuming and requires more effort but also takes up more space during archiving. The objective of the present study was to use commercially available different acrylic colors to ink specimen of appendix and fallopian tube of different patients, process them in a single cassette and preparation of single paraffin block and slide. Methods: 100 cases of appendix and fallopian tubes were taken to evaluate ink mapping technique using Fevicryl® Acrylic colours. Blocks and slides were prepared, labelling on the slides was done using the chromacodes method for identification of the specimen. These slides were then reported by the pathologist. Result: There was a 21% reduction in the amount of paraffin required for preparation of a double embedded block in comparison to two paraffin blocks by conventional method. It was also noted that there was 18% reduction in volume as compared to two conventional paraffin blocks. Conclusion: This method of using surgical inking technique not only brings down the effort and time required, but also storage space and the costs involved along with faster identification and reporting compared to the conventional method.

Improvised double-embedding technique of minute biopsies: A mega boon to histopathology laboratory

Optimal orientation of minute mucosal biopsies is essential for a definite diagnosis in gastrointestinal pathology or to visualize neural plexuses in Hirschsprung disease. The problem of minute size of the biopsy and its orientation gets compounded when they are from neonates and mandates exhaustive strip cuts, thus delaying reporting. A modified agar-paraffin technique is aimed to make tissue embedding efficient and user-friendly by inking mapping biopsies (one or more) either fresh or fixed with surgical coloring inks followed by embedding first in agar after orientation and followed thereafter by processing, re-embedding in paraffin wax, sectioning and staining. The tissues in agar paraffin block were found to be well processed, firm, held secure and well preserved. The blocks were easy to cut, with serial sections of thickness 2-3 μ and easy to spread. The colored inks remained permanently on the tissues both in the block as well as on the sections which helped in easy identification of tissues. Agar did not interfere with any stain such as Hematoxylin and Eosin or with histochemical stains, enzyme histochemistry or immunohistochemistry. Inking biopsies and pooling them in a block when obtained from the same patient reduced the number of tissue blocks. The modified agar-paraffin embedding technique is a simple reliable user friendly method that can greatly improve the quality of diagnostic information from minute biopsies by optimal orientation, better quality of sections, faster turnaround time and cost-effectiveness by economizing on the number of paraffin blocks, manpower, chemical reagents and laboratory infrastructure.

An improved double embedding technique for the microscopic evaluation of honey bees

An improved double embedding technique for the microscopic evaluation of honey bees

New double embedding technique for specimens of endoscopic submucosal dissection using agarose: comparison with other media

AimsProcedure of endoscopic submucosal dissection (ESD) has been introduced widely for treatment of early gastric cancers. For such specimens, accurate pathological diagnosis, especially concerning depth of the invasion and exposure...

The alveolar surface network: a new anatomy and its physiological significance.

It is generally held that the terminal lung unit (TLU) is an agglomeration of alveoli that opens into the branching air spaces of respiratory bronchioles, alveolar ducts, and alveolar sacs and that these structures are covered by a continuous thin liquid layer bearing a monomolecular film of surfactants at the open gas-liquid interface. The inherent structural and functional instability given TLUs by a broad liquid surface layer of this nature has been mitigated by the discovery that the TLU surface is in fact an agglomeration of bubbles, a foam (the alveolar surface network) that fills the TLU space and forms ultrathin foam films that 1) impart infrastructural stability to sustain aeration, 2) modulate circulation of surface liquid, both in series and in parallel, throughout the TLU and between TLUs and the liquid surface of conducting airways, 3) modulate surface liquid volume and exchange with interstitial liquid, and 4) sustain gas transfer between conducting airways and pulmonary capillaries throughout the respiratory cycle. The experimental evidence, from discovery to the present, is addressed in this report. Lungs were examined in thorax by stereomicroscopy immediately from the in vivo state at volumes ranging from functional residual capacity to maximal volume (Vmax). Lungs were then excised; bubble topography of all anterior and anterolateral surfaces was reaffirmed and also confirmed for all posterior and posterolateral surfaces. The following additional criteria verify the ubiquitous presence of normal intraalveolar bubbles. 1) Bubbles are absent in conducting airways. 2) Bubbles are stable and stationary in TLUs but can be moved individually by gentle microprobe pressure. 3) Adjoining bubbles move into the external medium through subpleural microincisions; there is no free gas, and vacated spaces are rendered airless. Adjacent bubbles may shift position in situ, while more distal bubbles remain stationary. 4) The position and movement of "large" bubbles identifies them as intraductal bubbles. 5) Transection of the lung reveals analogous bubble occurrence and history in central lung regions. 6) Bubbles become fixed in place and change shape when the lung is dried in air; the original shape and movement are restored when the lung is rewet. 7) All exteriorized bubbles are stable with lamellar (film) surface tension near zero. 8) Intact lungs prepared and processed by the new double-embedding technique reveal the intact TLU bubbles and bubble films. Lungs were also monitored directly by stereomicroscopy to establish their presence, transformations, and apparent function from birth through adulthood, as summarized in the following section. Intraalveolar bubbles and bubble films (the unit structures of the alveolar surface network) have been found in all mammalian species examined to date, including lambs, kids, and rabbit pups and adult mice, rats, rabbits, cats, and pigs. Rabbits were used for the definitive studies. 1) A unit bubble occupies each alveolus and branching airway of the TLU; unit bubbles in clusters correspond with alveolar clusters. 2) The appositions of unit bubble lamellae (films) form a network of liquid channels within the TLUs. The appositions are bubble to bubble (near alveolar entrances, at pores of Kohn, and between ductal bubbles), bubble to epithelial cell surface, and bubble to surface liquid of conducting airways. They rapidly form stable Newtonian black foam films (approximately 7 nm thick) under hydrodynamic conditions expected in vivo. 3) Lamellae of the foam films and bubbles tend to exclude bulk liquid and thus maintain near-zero surface tension. At the same time, the foam film formations--abetted by the constant but small retractive force of tissue recoil--stabilize unit bubble position within the network. 4) Unit bubble mobility in response to applied force increases as liquid accumulates within the network (e.g. (ABSTRACT TRUNCATED)

Determination of the 3-D morphology of degradable biopolymer implants undergoing in vivo resorption.

A technique is presented that allows the visualization of the degradation process of biopolymers. Serial thick-section specimens containing cross-sections of polydioxanone implants (PDAs) were realigned for 3-D reconstruction using a double-embedding technique. The outlines of the cross-sectioned implants were traced using an automatic image analysis system and converted into x, y, and z coordinates. The reconstruction of the implant body was achieved by the stepwise analysis of vertical relationships between two adjacent section levels. The surface was approximated by triangulation and calculated from the surface triangles. Visualization was achieved by introducing the data into commercially available software using z-buffering and Gouraud shading. The geometric precision of the reembedding technique was found to be 0.8-1.5% of the screen width; recovering experiments showed a good approximation to the actual physical volume of unresorbed implants (+/- 2.6%). The error of volume determination due to the limited resolution in the z direction was calculated to be acceptable (< 5.0%) in isotropic objects where the ratio between the radius of the surface curvature and the distance between the sections is > 4. Results indicate that large devices of PDAs are degraded in vivo in a fashion similar to that previously described for in vitro degradation of PLA molded screws by a surface/center differentiation with formation of hollow residuals after 17 weeks and complete degradation with phagocytosis of microparticles after 26 weeks.

A double-embedding technique for thin tissue membranes.

A new double-embedding technique for thin tissue membranes is presented. This technique is useful for thin membranes such as mesenteric membranes from rodents, which usually measure only 10 microns in thickness. Several membranes are fixed and mounted on four needles located at the bottom of a plastic box. The box is filled with agarose at 50 C and then allowed to solidify. The agarose block is then removed, dehydrated in alcohol, cleared with HistoPetrol (isoparaffin hydrocarbons), permeated with paraffin and sectioned. The morphology is comparable to that obtained with methacrylate plastic embedding but is less time-consuming, less hazardous since no plastic hardener and activator are used and makes immunohistochemical studies easier.

Immunocytochemical localization of water-soluble glycoproteins, including Group 1 allergen, in pollen of ryegrass,Lolium perenne, using ferritin-labelled antibody

The cellular sites of the glycoproteins Group 1 allergen (glycoprotein 1) and Antigen A (glycoprotein 2) in mature ryegrass pollen have been investigated by immunoelectron microscopy. Radioimmunoassays confirm previous findings of cross-reactivity between the purified glycoprotein antigens at the high immunoglobulin G (IgG) concentrations used for localization. Freeze-drying of anthers followed by anhydrous processing has been employed because of the water solubility and mobility of the glycoproteins. A double-embedding technique has been developed. This involves, first, embedding anthers in the water-soluble plastic resin JB-4, sectioning and incubating in ferritin-labelled antisera by the indirect method. The sections are then embedded in Spurr's resin for ultra-thin sectioning. Both glycoproteins are found in the following sites: (1) exine and intine wall layers; (2) pollen cytoplasm; (3) the orbicules and anther loculus; and (4) the anther cuticle. In the exine arcades and surface and in the anther loculus, the ferritin label is bound to pollenkitt. The finding that the glycoproteins are in similar sites is predictable in view of the cross-specificity of the antisera. The extent of antibody penetration of the plastic sections has been examined; labelling is confined to cut grains and absent from intact grains.

A Double Embedding Technique For The Electron Microscopic Analysis Of The Liver Acinus

A double embedding technique allowing accurate and systematic sampling of zones of the hepatic acinus by electron microscopy was developed. The method involved the primary embedding of large (10 mm x 30 mm x 0.5 mm) liver slices and the preparation of 10 micrometers thick tissue sections by means of a rotary microtome with a steel knife. Veins 20--40 micrometers in diameter, the landmarks of the hepatic acinus, were localized by light microscopy, dissected from surrounding tissue and reembedded for ultramicrotomy. The method facilitated the systematic evaluation of hepatocyte ultrastructure in each zone of the microcirculatory unit of the liver, the hepatic acinus.

Bromobenzene-induced zonal necrosis in the hepatic acinus

The time course and acinar distribution of bromobenzene-induced hepatic necrosis was studied in the rat. Cellular damage, lipid infiltration, and changes in glycogen deposits were investigated by light microscopy 6, 16, 24, and 48 hr after bromobenzene (BZ) administration. Concomitantly, ultrastructural changes were followed by electron microscopy in each zone of the acinus. To insure accurate orientation in acinar zones, a double embedding technique for electron microscopy was used. Acinar zones were localized by light microscopy and subsequently re-embedded for electron microscopy. Zone 3 was the site of conformational changes in smooth endoplasmic reticulum 6 hr after BZ administration. This condensed, tubular network represented the earliest morphological sign of injury observed by electron microscopy. At 48 hr, cytoplasmic vacuolar degeneration and necrosis were observed in the hepatocytes of acinar zone 3. While no necrosis was observed in the cells of zone 1, other morphological changes occurred. These included progressive lipid accumulation, as well as fluctuations in the amount of rough endoplasmic reticulum and free ribosomes. These latter observations suggested a possible link between protein manufacture and survival of the zone 1 cells. These results established that, following bromobenzene administration, necrosis was restricted to zone 3 hepatocytes.

Electron microscope studies of the microvilli of HeLa cells.

Microvilli of HeLa cells cultured in vitro were preserved for electron microscopic examination at different stages of routine cultivation procedures. By a double-embedding technique, vertical sectioning for electron microscopy was possible. It revealed that, although the microvilli were present on all sides of the cell in the dispersed stage and in the attached stage, they were not present on the bottom of the cell when it was stretched on the surface of the dish. When the cells were grown in dense colonies, they were found on top of each other, and microvilli were present on all sides, except on the bottom surface of those cells in contact with the dish. We achieved a more dramatic demonstration of the microvilli by developing a surface-replica technique which retains their spatial arrangement and permits characterization of the distribution of their number, length, and diameter.

Serial section radioautography of the inner ear. Histological technique.

THE FOLLOWING histological technique was developed in order to obtain routine serial sections of mouse temporal bones of a quality suitable for optimal light microscopy and radioautography. Light microscopic radioautography with tritium requires that sections be cut no thicker than 10μ and that they lie flat on the slide. These criteria are not readily met in the case of the heterogeneous tissues of the inner ear. A double embedding technique 1 was discarded because too long a time was required. Paraffin embedding, used by other investigators, 2-4 has resulted in poor cytological detail and artifacts in the radioautographs. Frozen sections of tissues embedded with polyvinylpyrrolidone (PVP) or gelatin were studied but the sections did not lie flat, it was difficult to obtain serial sections, and the cochlea sections usually fragmented. The method to be described is similar to one reported earlier, 5 but with modifications that were found helpful in

Wool fibres: sectioning and staining, differentiation of ortho and paracortex.

A double embedding technique for tangential sectioning of hair and wool fibres is as follows: The cleaned fibre bundle is attached to a U-shaped, 16 gauge, tinned-copper wire frame with collodion adhesive, soaked in 6% nitrocellulose for 1 hr, and treated with chloroform for 2 hr. The hardened bundle is then cut fom the wire support and embedded in paraffin-beeswax, 95:5. Sectioning is at 6-8 μ. The use of 2% orange G or saturated aqueous picric acid for quantitative study of the fibres, and the demonstration of wool fibre cortical fractions by staining with polychrome methylene blue after oxidation of the sectioned fibres in a solution of formic acid (98/100 w/v) 25 ml; distilled water, 65 ml; and H2O2 (30% w/v), 10 ml, for 1 hr, is recommended.

Tetrahydrofuran and its Use in Insect Histology

Tetrahydrofuran (tetramethylene oxide) has been recommended as a histological dehydrating and clearing agent for use in human pathology by Dahme and Rothemund (1) and more recently by Haust (2). Dahme and Rothemund recommended its use after a search for a non-toxic reagent miscible with both water and molten paraffin wax. These authors state that the toxicity of tetrahydrofuran only approaches that of ethyl ether, whereas dioxane, with similar properties, is highly toxic. Tetrahydrofuran would appear to be as safe for routine laboratory use as toluene and xylene when similar precautions are observed. Haust (1958) gives various schedules for its use on human pathological material, and compares it with the more traditional methods. She concludes that it is simpler to use, and at least equally as efficient as the conventional dehydrating and clearing agents. In our work on insects we have extended its use to a solute for fixing solutions, and for dissolving celloidin in a double embedding technique.