Histology and Histochemical Stains
I. RECEIPT, ACCESSIONING, AND GROSS DISSECTION. Most, if not all, biopsies and large tissue specimens are routed to the pathology laboratory. Under normal circumstances, the specimens are received in 10% neutral buffered formalin (formalin begins the fixation process and prevents autolysis and decomposition). The specimen is first logged into the surgical pathology computer system and given a unique identifying number, referred to as an accession number or case number. Once accessioned, the specimen is taken to the gross dissection room; depending on the practice setting, residents, fellows, pathologist assistants, and/or trained technicians are responsible for gross processing of the specimen under the supervision of an attending pathologist. Gross processing entails describing the specimen by its size, shape, color, and overall general appearance, followed by placing samples of the tissue in processing cassettes (for biopsies, the entire tissue specimen is placed in a cassette; for larger specimens, regions of tissue are sampled according to established protocols). Each cassette is labeled with the accession number as well as a part designator and number; this numbering scheme is designed to allow the location of a particular section of tissue within the context of the whole specimen.
II. PROCESSING. The loaded cassettes are stored in 10% neutral buffered formalin until automated tissue processing. The normal processing cycle is ˜8 hours long and in general is designed to remove the water from the specimen and replace it with paraffin. Automated, closed-system tissue processors utilize agitation, vacuum, and increased temperature to optimize the process. In general terms, the process is as follows. First, the tissue is subjected to 10% neutral buffered formalin to ensure complete fixation. Complete fixation aids in the dehydration steps and prevents tissue shrinkage and other artifacts caused by excessive or rapid dehydration; chemically, formalin fixation produces methylene cross-links between nucleic acids and/or proteins. Once the tissue is well fixed, it is subjected to several changes of graduated alcohols in a gradient starting at 70% and ending at 100%, a process that removes water from the tissue at a slow controlled rate designed to prevent excessive shrinkage and disruption of the architecture and cellular components. After complete dehydration of the tissue has been accomplished, a clearing agent is used to remove the alcohol and allow tissue infiltration by paraffin; this clearing agent must therefore be miscible in both alcohol and paraffin. Xylene is most often used for this purpose, although commercial xylene substitutes are available. In the next step of processing, heated paraffin infiltrates into the tissue. Paraffin is a solid at room temperature but has a relatively low melting point, and so is a good choice as an infiltration and embedding media. While pure paraffin wax was used in the past, current commercially available paraffins are formulated with various plastic polymers to allow better infiltration and a more rigid crystalline structure, both of which aid subsequent microtomy.
III. EMBEDDING. Properly fixed and processed tissue sections are embedded in molds to prepare them for microtomy. The tissue is removed from the cassette and oriented in the base of a mold that is of a size to allow paraffin to surround the tissue section. During embedding, the tissue is oriented with the understanding that the surface placed down in the mold will become the face of the tissue block, and will thus be the surface cut into first by the microtome blade. Attention must be
given to tissues requiring specific orientation such as tubular structures requiring complete cross sections (e.g., ureteral margins). Orientation is critical to proper tissue representation on the finished slide and leads to proper pathologic diagnosis, and so the importance of proper embedding cannot be overstated. After proper orientation, the mold and tissue are touched to a cold plate to begin to solidify the paraffin so that the tissue is held in place as the mold is filled with paraffin. The empty cassette is placed on top of the mold so that it becomes the back of the tissue block, conveniently retaining the identification of that tissue sample. Finally, the mold is allowed to cool so that the paraffin block containing the oriented tissue can be easily removed.
IV. MICROTOMY. Proper microtomy requires a well-trained and highly skilled microtomist, usually a trained histotechnician or histotechnologist. The microtome instrument is designed to hold the paraffin tissue block firmly in place as it is cyclically presented to a stationary microtome blade. Each turn of the microtome handle (each cycle) advances the tissue block a set distance, so microtomes must be kept clean and in good working order. Most tissues are sectioned at 4 to 5 µm thick; however, some tissues are cut thinner at 3 µm (e.g., kidney biopsies and lymph node biopsies), and others thicker at 5 to 6 µm (e.g., bone and brain).
In practice, the paraffin block is first “faced in” to reach a level within the tissue where there is a representative tissue section plane; the block is then cooled on wet ice to further harden the paraffin and aid microtomy. If proper care is taken, individual sections come off of the microtome blade connected to each other, a string of tissue sections called a “ribbon.” The tissue ribbon is then floated on a warm water bath at a temperature 6°C to 8°C below the melting point of the paraffin to make the paraffin very pliable. This aids in mounting the sections on a glass microscope slide labeled with the corresponding accession number and part identifier for that particular block (in addition, the convection currents formed in the water bath help gently stretch the tissue sections, removing any wrinkles).
V. HEMATOXYLIN AND EOSIN (H&E) STAINING. Slides that have been sectioned are stained to reveal their histologic detail. The primary stain used for pathologic diagnosis is the H&E stain. Hematoxylin is derived from the log wood tree, and has long been in use in the pathology laboratory. By itself it is not a dye, but once it is oxidized to hematein and combined with a metallic mordant, it acquires a strong affinity for nuclear chromatin. Eosin is a dye that at a pH of approximately 4.6 to 5.0 is a strong anion and thus has an affinity for positively charged, cationic, tissue protein groups. At the proper pH, eosin combines at different rates to tissue proteins and thus produces a graduation of distinct shades from light pink to pinkish red.
There are several methods for performing the H&E staining that can be used to achieve slight variations in the end stain to match the preference of the pathologist. The two general variations of H&E staining in common use are progressive and regressive methods. Progressive methods involve staining slides for a designated period of time, and then stopping the reaction as soon as optimal staining has occurred; every tissue stains slightly differently with hematoxylin on the basis of its type, fixation, and prior decalcification, and therefore the length of time in hematoxylin is critical using the progressive method. Regressive methods overstain the tissue sections with hematoxylin, and then differentiate the hematoxylin using acid alcohol; by overstaining and then differentiating the hematoxylin by the regressive method, a darker, crisper stain can be achieved with the assurance that all hematoxylin-positive elements are represented.
The routine regressive staining protocol for the H&E stain is briefly as follows. The tissue sections are dried completely in an oven since water left on or under the tissue sections can allow the sections to fall off of the slide during the staining process. The slides are then deparaffinized by soaking in xylene, the xylene is removed by alcohol, and the slides are rehydrated by 95% alcohol and then water before staining with hematoxylin. Excess hematoxylin is then removed with a water
rinse, the slides are differentiated using acid alcohol, rinsed, and the hematoxylin is “blued” by immersion in a weak ammonia water solution. The slides are rinsed again, placed in 80% alcohol, and stained with eosin. Excess eosin is removed by alcohol rinses, and the slide is prepared for mounting with a coverslip and resinous media by removal of the alcohol using xylene rinses.
VI. OTHER FREQUENTLY USED HISTOCHEMICAL STAINS. Tissue stains range from very simple to complex in methodology, and can be used to demonstrate most major tissue elements relevant to pathologic diagnosis. They are based on the chemistry of various dyes and metals, and most were developed prior to the advent of immunohistochemistry. In general, a histochemical stain consists of the main chemical reaction that demonstrates the specific tissue element of interest, followed by chemical reactions that provide staining of the background uninvolved tissue elements, often including nuclear detail. Histochemical stains are usually grouped by the tissue element they stain.
A. Carbohydrates. In humans, carbohydrates exist as various sugars and polymers linked to proteins. Simple sugars cannot be detected by standard histochemical procedures because they are water soluble and thus removed during processing; however, polymers such as glycogen can be detected. Naturally occurring polysaccharides can be classified into four groups on the basis of their histochemical staining differences: neutral polysaccharides (Group I), acid mucopolysaccharides (Group II), glycoproteins (Group III), and glycolipids (Group IV). Amyloid must also be included because, even though it is not a carbohydrate, its histochemical staining properties are similar to those of polysaccharides. The histochemical stains most often used to detect carbohydrates and differentiate various types of carbohydrates are Alcian blue, colloidal iron, mucicarmine, the periodic acid-Schiff (PAS) reaction, Congo red, and Thioflavin T.
1. Mucicarmine. The mucicarmine method is used to detect tissue mucins and utilizes the tissue dye carmine. When carmine is reacted with aluminum, it forms a compound that has a net positive charge and is attracted to the negative acid groups of epithelial mucins. Metanil Yellow and Weigert’s hematoxylin are used as counterstains and produce yellow staining of the background tissue elements and blue-black nuclear staining (e-Fig. 54.1).*
2. Alcian blue, a phthalocyanine basic dye, forms salt bridges with the acid groups in mucopolysaccharides. Staining tissue sections in an Alcian blue solution at pH 1.0 produces staining of only sulfated mucopolysaccharides, while staining at pH 2.5 produces staining of all mucopolysaccharides. These two methods make it possible to differentiate sulfated from carboxylated mucopolysaccharides; further differentiation between mucosubstances of connective tissue origin and that of epithelial origin can be achieved by the addition of hyaluronidase digestion (e-Fig. 54.2).
3. Colloidal iron. The colloidal iron stain is based on the chemical principle that at low pH colloidal ferric ions can be absorbed by both carboxylated and sulfated mucopolysaccharides, as well as other glycoproteins. The absorbed ferric ions are detected by use of the Prussian blue reaction (see below).
4. Congo red. Congo red reacts with cellulose and amyloid. The dye is a linear molecule that attaches to amyloid in a sheet-like fashion resulting in so-called apple green birefringence when subjected to polarized light. This “apple green” birefringence is considered specific for amyloid in Congo red-stained tissue sections (e-Fig. 54.3).
5. Thioflavin T. Thioflavin T is a fluorescent tissue dye that has an affinity for amyloid. Thioflavin T fluoresces yellow to yellow-green when the tissue
section is viewed by fluorescent microscopy, but the dye is not as specific for amyloid as Congo red.
6. PAS. The PAS reaction is invaluable in histochemistry because of its versatility. In this reaction, the glycol groups of polysaccharides, mucosubstances, and basement membranes are subjected to oxidation by a solution of periodic acid. The oxidation of the glycols results in the formation of dialdehydes. The dialdehydes are then reacted with Schiff’s reagent, a colorless solution created by reducing basic fuchsin in the presence of sulfurous acid. When reacted with the previously oxidized tissue, Schiff’s reagent is bound to the dialdehyde groups and gains a red color (e-Fig. 54.4).
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