Histology and Toxicology Questions 622555 1) Haematoxylin is a natural substance used in tissue staining.

The Haematoxylin itself isnt an adequate material for staining. It first needs to be converted into Haematein by natural oxidation or chemical oxidation. Natural oxidation is carried out by exposing the Haematoxylin to light and air. This is a slow process, sometimes taking as long as 3-4 months, but the resultant solution seems to retain its staining ability for a long time (Wilson & Gamble, Theory and Practice of Histological Techniques 2002). Chemical agents oxidise haematoxylin into haematein almost immediately, however, the longevity of the stain is often short lived. Chemical oxidation in Harriss Haematoxylin was originally achieved by using the compound mercuric oxide, although due to the toxicity of it, sodium or potassium iodate are used in modern Haematoxylin staining. Haematein however, is anionic, meaning that it has a poor affinity for tissue. This issue is alleviated by utilising salts of aluminium, iron and tungsten, which act as a mordant, the combination of dye and mordant is known as a lake. The inclusion of a metal cation bestows the mordant-dye lake with a net positive charge. This net positive charge enables anionic tissue such as nuclear chromatin to bond to the mordant-dye lake. The use of different mordants yields varying coloured stains and determines the tissue in which the stain adheres to.

2) Acid alcohol is used to remove any excess stain from a tissue sample during haematoxylin and eosin staining. It is extremely efficient at this, as it is very easy to selectively strip the tissue sample of dye using this method. This happens because the acid simultaneously causes an increase in the ionisation of amino groups and a reduction in the ionisation of carboxyl and hydroxyl groups within the dye-mordant complex and the tissue. This causes the dye-mordant complex to disassociate with the tissue and dissolve into the acid. This is done by ensuring the acid (usually hydrochloric) has a stronger affinity with the cationic dyemordant complex than the anionic tissue its bound to. This shifts the colour of the staining from blue to pink. After this a blueing agent such as Scotts tap water substitute is applied to re-establish the blue/purple colour of the stain. This happens because the slightly basic solution re-establishes the insoluble dye lake. 3) Histology commonly uses Formaldehyde in some form as a fixative as it is inexpensive, easily available, fairly convenient to store, relatively safe and retains fairly competent morphology. It is used as a fixative by making an aqueous solution of 9 parts water to 1 part strong formalin containing 40% formaldehyde. When formaldehyde comes into contact with protein it causes it to create linkages that form a mesh that retains morphology. Methylene bridges are formed between 2 side amino groups of glutamine and lysine on separate protein chains. This is an oxidative reaction and is shown in the diagram below.

Another fixative used in histology is a compound called Bouin solution. Bouin solution is an aqueous solution composed of picric acid, acetic acid and formaldehyde. The functions of the 3 chemicals in Bouin solution complement one another. Cytoplasm becomes basophilic and the tissue is also hardened because of the formalin but this is countered by the effect of the picric acid. The swelling effect of acetic acid upon the tissue is countered by the shrinking effect picric acid has upon tissue. All of these things result in better Haematoxylin and eosin staining and clearer nuclear and cytoplasmic staining. Acetic acid within Bouins solution lyses red blood cells and dissolves small iron and calcium deposits in tissue because of this it is often used for trichrome staining. 4) Tissue processing is used to embed tissue into a media strong and rigid enough to protect and support the tissue sample but yet be soft enough to not damage the tissue sample and not cause damage to cutting instruments. The usual media used for this is paraffin wax. Paraffin wax is used because most fixatives are aqueous based and are not miscible with it. There are 3 stages involved in tissue processing they are dehydration, clearing and embedding. Dehydration: This stage removes water and fixative from the tissue sample and replaces it with dehydrating fluid. Reagents that are used to achieve this are ethanol, methanol, propan-2-ol isopropyl alcohol and acetone. Some of these reagents work by attracting water from the tissue because of their hydrophilic properties and others work by progressive dilution of the aqueous tissue fluids. Clearing: As most dehydrating agents arent miscible with paraffin wax they need to be replaced by an agent that is both miscible with the paraffin wax and the dehydrating agent, this is known as clearing. These reagents are usually hydrocarbon solvents with refractive indices comparable to protein. Once the dehydrating agent has been completely replaced by the clearing agent it will have a translucent appearance. Reagents used for this are xylene, toluene, chloroform, paraffin, citrus fruit oils, methyl benzoate and methyl salicylate. Embedding: The last stage consists of embedding the tissue sample in paraffin wax. This is done by dispensing hot paraffin into a mold and adding the tissue sample. The wax is then placed onto a cooling plate so as to set quickly. The sample is then ready for sectioning.

5) The Periodic Acid Schiff method or PAS method is a histological stain used to identify carbohydrates in tissue samples. The carbohydrates involved are 1-2 glycols. Carbohydrates dont all contain these structures however, so the PAS method is not used for all carbohydrates but only for those containing 1-2 glycols. These include polysaccharides, mucopolysaccharides, glycolipids and glycoproteins. For this stain to work it is essential that the carbohydrates produce an aldehyde. This is done by oxidising the carbohydrates 1-2 glycols with periodic acid as shown below.

After this Schiff reagent is added which forms a strong magenta compound with aldehydes when exposed to them. This reaction is shown below.

In Kidney tissue stained with this method cuboidal epithelial cells and low epithelial cells are stained. The basement membrane is dyed bright magenta and the glycocalyx brush border is also stained. The basophilic nuclei are stained blue in colour with the basic blue component of the stain, while the acidophilic cytoplasm of these cells is dyed a pink colour, being stained by the red acid part of the stain. 6) Massons a histological staining method is a trichrome stain. The principle of this stain is to identify and differentiate between collagen fibres, fibrin, and erythrocytes using 3 different dyes. Trichrome staining works by the smaller molecules of dye staining the most porous tissue and the larger dye molecules staining the less porous tissue. If a large molecule of dye can work its way into a tissue it will do it at the expense of smaller molecules of dye. In this method Nuclei are stained blue/black, muscle and erythrocytes red and collagen blue. 7) Connective tissue cells have many functions within the body and are distributed throughout it. The functions of connective tissue include Binding and support, Protection, Mineral Storage, Insulation, Energy Storage, Transportation (blood) and Immunity. Some examples of connective tissue cells are listed below.

Fibroblasts: Fibroblasts are the most predominant type of cell of the connective tissue. Fibroblasts produce the vast majority of the extracellular matrix and are found all over the body. Adipose cells: Adipose or fat cells are spherical and large. The main volume of the cell is made up of a droplet of fat. Fat cells dont move or divide. Sympathetic neurotransmitters, insulin and glucagon receptors are positioned within the plasma membrane of the cell. Adipose cells are responsible for storing fat deposits and the autonomic nervous system is responsible for regulating the moving of fat from the cells and into the cells. Another form of adipose tissue is brown fat. Brown fat cells can quickly metabolize their own lipids to make heat. Brown fat contain many mitochondria which give it its colouration. Mast cells :Mast cells are all over the body. They are used in defence of the body. They work by stimulating other connective tissues. This happens because they degranulate, producing granules. Heparin-protein complex, histamine, phospholipid, anaphylactic factors, and basic proteins are within these granules. These products stimulate other connective tissue in order to protect the body. Macrophages: Macrophages have 3 main responsibilities. These are the collection of waste, storage of waste and defence. They defend the body by ingesting and destroying bacteria and fungi. There many different forms of macrophage. There exist different populations of macrophage. These include: alveolar macrophages in the lung, peritoneal macrophages in the peritoneal cavity, synovial macrophages in the synovial cavity, Kupffer cells in the liver, and histiocytes in the connective tissue proper.

8) Connective Tissue 3770 Photo by: Alila

The human body is composed of just four basic kinds of tissue: nervous, muscular, epithelial, and connective tissue. Connective tissue is the most abundant, widely distributed, and varied type. It includes fibrous tissues, fat, cartilage, bone, bone marrow, and blood. As the name implies, connective tissues often bind other organs together, hold organs in place, cushion them, and fill space. Connective tissue is distinguished from the other types in that the extracellular material (matrix) usually occupies more space than the cells do, and the cells are relatively far apart. Fat is an exception, having cells in close contact with each other; but with large, nonliving, intracellular lipid droplets, fat contains much more nonliving material than living material.

The matrix of connective tissue typically consists of fibers and a featureless ground substance. The most abundant fiber in connective tissues is a tough protein called collagen. Tendons, ligaments, and the white stringy tissue (fascia) seen in some cuts of meat are composed almost entirely of collagen, as is leather, which consists of the connective tissue layer (dermis) of animal skins. Collagen also strengthens bone and cartilage. Elastic and reticular fibers are less abundant connective tissue proteins with a more limited distribution.

The ground substance may be liquid, as in blood; gelatinous, as in areolar tissue; rubbery, as in cartilage; or calcified and stony, as in bone. It consists mainly of water and small dissolved ions and organic molecules, but the gelatinous to rubbery consistency of some tissues results from enormous protein-carbohydrate complexes in the ground substance. The hard consistency of bone results mainly from calcium phosphate salts in the ground substance. The principle of staining reticulin with ammoniacal silver solution is to recognise tissue by selectively depositing metal onto the tissue. This is done by first oxidising the tissue, this is usually done with permanganic acid. This is done by the mixing of potassium permanganate and sulphuric acid. This decreases the amount of silver solution that binds to other tissue making the reticulin have a greater contrast. Next a solution of oxalic acid is added to bleach the brown discolouration of the potassium permanganate.

9) 1.Mild oxidation of the tissue, often with permanganic acid made by mixing potassium permanganate and sulphuric acid, or with potassium permanganate alone. It is possible that this oxidation step produces binding sites on the fibre and makes the reticulin fibres more reactive to the sensitiser. That is not a very satisfactory statement and makes the explanation somewhat ill defined, but it is not possible to be more definitive that that. It is also possible that this oxidation decreases binding sites on tissues other than reticulin and, as a consequence, also decreases the amount they reduce the silver solution, causing the impregnation of reticulin to have greater contrast in comparison. It could also be that both processes are taking place at the same time. 2.Bleaching the discolouration produced by the permanganate, usually with an aqueous solution of oxalic acid, which is also a mild oxidising agent. It should be noted, however, that there is no suggestion that the oxalic acid further oxidises tissue components and it is applied solely to remove the brown discolouration produced by the permanganate. 3.Treating with a "sensitiser". Often this is iron alum (ferric ammonium sulphate), although other sensitisers, including silver nitrate, have also been used. It is not clear what happens, but the commonest explanation is that the (ferric) iron combines with binding sites freed up by the permanganate oxidation and attaches to the reticulin fibre predominantly, with much lower amounts attaching to other components. This iron alum is then washed off, but only for long enough to remove the excess. Too much washing removes it from the reticulin fibres and results in a poorer quality impregnation. 4.Applying ammoniacal silver solution for a short time, usually about thirty seconds. It is thought that the silver compound attaches to the reticulin fibres in both a focal and nonspecific fashion. The focal attachment is to the iron bound to the fibres, and the non-specific

attachment is to the reticulin fibres in general, although the specifics are never spelled out. There is also some non-specific attachment to the tissue in general. The excess ammoniacal silver solution is then rinsed off sufficiently to ensure there is no free silver compound remaining and the only silver salts present are bound to the tissue, but not for long enough to remove any of the bound silver salts. At this stage most of the silver compound is not reduced, but isolated foci of reduced silver may be present, perhaps at the locations where the iron attached during sensitising. At this stage the tissues often have a very pale, light brown, slightly translucent appearance. 5.Reducing with a chemical reducing agent, usually 10% formalin in tap water. It is important that formal saline or neutral buffered formalin not be used as the buffer salts and sodium chloride interfere with the reduction. Plain formalin diluted with water is needed. This chemically reduces the silver attached to the tissue. Since most of it is attached to reticulin fibres, these are darkly impregnated on a much paler background. It is thought that the silver attached focally in association with the iron may act as nodes for the reduction in much the same way as light sensitised silver halide atoms in photographic film act as nodes for reduction of the whole silver halide grain during development with photographic developers, which are chemical reducing agents. 6.Following chemical reduction with formalin, excess chemicals are removed and the impregnation stabilised with water washes or treatment with sodium thiosulphate. 10) Background

Special stains, such as reticulin stain and CD34 immunostain, are very helpful in the diagnosis of well differentiated hepatocellular carcinoma (HCC). Most studies have shown that absent or decreased reticulin stain or an abnormal reticulin pattern with widened trabeculae is reliable for the diagnosis of well-differentiated HCC.

Case report

We report here two cases of well differentiated HCC with an unusual reticulin staining pattern. A strongly positive reticulin network was preserved within the tumor, which surrounded individual tumor cells in a monolayered trabecular pattern. At the same time, an increased CD34 stain was present in the tumor.

Conclusions

This unusual reticulin pattern represents part of the diverse reticulin staining patterns seen in HCC. Although this staining pattern is rare, it should be recognized when diagnosing well-differentiated HCC in small samples such as cellblock of fine needle aspiration or small core biopsies.