Pigments and minerals

Normal and abnormal iron metabolism

Dietary iron is absorbed in the small intestine and attached to a protein molecule for transfer to the sites in the body where it is to be utilized or stored. Approximately 30% is stored within the reticuloendothelial system, especially the bone marrow. The bone marrow is the main site of iron utilization in the body and where it is incorporated into the hemoglobin molecule during red cell formation. The normal breakdown of worn-out red cells results in the release of iron which is recirculated back into the various areas of iron storage for further utilization. Under normal conditions, this efficient system of recycling usually means that iron deficiency rarely occurs. There is a minimal loss of iron by the way of epithelial desquamation, hair loss and sweating. The main reason for loss of iron from the body is hemorrhage in the form of either chronic bleeding (e.g. a peptic ulcer or bowel neoplasia), or in the female by menstruation, with approximately 25% of females being iron deficient. The small intestine normally only absorbs sufficient iron from the excess iron in the diet to counteract any losses, but in excessive blood loss the dietary content may be relatively inadequate for the need, and even though the absorption mechanism is working at full efficiency, a state of clinical iron deficiency occurs. In iron deficiency, the iron stores in the bone marrow become depleted, insufficient hemoglobin is produced because of the lack of iron, and anemia develops in which the red cells contain diminished amounts of hemoglobin. The iron deficiency is characteristically demonstrated by the absence of stainable iron in the bone marrow.

There is no active method of iron excretion from the body. Iron excess is a much less common condition; under normal conditions the intestine will not absorb iron from the diet when there is already a surplus within the body. However, this controlling mechanism may be bypassed when iron is given therapeutically, such as in the form of either iron injections or a blood transfusion. If excess iron is given this way, the iron stores may become overloaded, and excessive amounts of hemosiderin may be deposited in the organs with a prominent reticuloendothelial component e.g. spleen, bone marrow or liver. This condition is called hemosiderosis . A rarer cause of iron overload is the genetic disease hemochromatosis , in which the controlling mechanism at the small intestine absorption stage becomes impaired, and the iron is absorbed indiscriminately in amounts irrelevant to the state of the body’s iron stores. In this disorder, large quantities of hemosiderin are deposited in many of the organs, often interfering with those organs’ structure and function.

Demonstration of hemosiderin and iron

In unfixed tissue, hemosiderin is insoluble in alkalis but freely soluble in strong acid solutions. After fixation in formalin, it is slowly soluble in dilute acids, especially oxalic acid. Fixatives which contain acids but no formalin can remove hemosiderin or alter it in such a way that reactions for iron are negative. Certain types of iron found in tissues are not demonstrable using traditional techniques. This is because the iron is tightly bound within a protein complex. Both hemoglobin and myoglobin are examples of such protein complexes and, if treated with hydrogen peroxide (100 vol), the iron is released and can then be demonstrated using Perls’ Prussian blue reaction ( Fig. 14.1 ). A similar result is obtained if the acid ferrocyanide solution is heated to 60°C in a water bath, oven or microwave oven. However, the use of heat will sometimes cause a fine, diffuse, blue precipitate to form on both the tissue section and slide. This precipitate will not occur when the slides are stained at room temperature. Metallic iron deposits, or inert iron oxide seen in tissues because of industrial exposure, are not positive when treated with acid ferrocyanide solutions. As a consequence of the tissue response, various mechanisms release some of the iron in a demonstrable form, and such deposits are almost invariably surrounded by hemosiderin. In almost all the instances where demonstrable iron appears in tissues, it does so in the form of a ferric salt. On those rare occasions when iron appears in its reduced state as the ferrous salt, then method may be used to achieve the Turnbull’s blue reaction to visualize its presence in tissue sections ( Fig. 14.2 ).

An interesting and sometimes useful modification of a serum iron technique was introduced by to demonstrate both ferrous and ferric iron in tissue sections. This method was claimed to be a more sensitive demonstration for the detection of both ferric and ferrous salts, but has not succeeded in replacing the more traditional method for the demonstration of iron. The method uses bathophenanthroline and the resultant color of any iron present in tissues is bright red.

Demonstration of hemoglobin

Two types of demonstration method can be used to stain hemoglobin in tissue sections. The first demonstrates the enzyme, hemoglobin peroxidase, which is reasonably stable and withstands short fixation and paraffin processing. This peroxidase activity was originally demonstrated by the benzidine-nitroprusside methods, but because of the carcinogenicity of benzidine these methods are not recommended and are no longer used. introduced the patent blue method which was later modified by ( Fig. 14.3 ). Tinctorial methods have also been used for the demonstration of hemoglobin; the amido black technique ( ) and the kiton red-almond green technique ( ) are worth noting.

Demonstration of bile pigments and hematoidin

The need to identify bile pigments arises mainly in the histological examination of the liver, where distinguishing bile pigment from lipofuscin may be of significant importance. Both appear yellow-brown in H&E-stained paraffin wax sections, and it is worth remembering that the green color of biliverdin is often masked by eosin. In such cases, unstained paraffin wax or frozen sections, lightly counterstained with a suitable hematoxylin (e.g. Mayer’s), will prove of value. Bile pigments are not autofluorescent and fail to rotate the plane of polarized light (monorefringent), whereas lipofuscin is autofluorescent. The most commonly used routine method for the demonstration of bile pigments is the modified Fouchet technique ( ), in which the pigment is converted to the green color of biliverdin and blue cholecyanin by the oxidative action of the ferric chloride in the presence of trichloroacetic acid ( Fig. 14.4 ). The Fouchet technique is quick and simple to carry out, and when counterstained with van Gieson’s solution the green color is accentuated.

Oxidation methods aim to demonstrate bilirubin by converting it to green biliverdin. In practice they fail to produce the bright blue-green color seen in the more popular Fouchet technique and tend to be a dull olive green color. These oxidation methods are of little value in routine surgical pathology and are rarely used. Another group of methods which have been used to demonstrate bile pigments are the diazo methods which are based on a well-known technique previously used in chemical pathology, namely the van den Burgh test for bilirubin in blood. The method is based on the reaction between bilirubin and diazotized sulfanilic acid. modified the method for use on cryostat sections but the reagents are complex to make up and section loss may be high, therefore, its use is limited.

The most common sites where melanin can be found are

• 1.

  Skin , where it is produced by cells called melanocytes which are usually scattered within the basal layer of the epidermis. In certain inflammatory skin diseases melanin may also be found in phagocytic cells (‘melanophages’) in the upper dermis. The melanophages may also phagocytize other material such as lipofuscins and lipoproteins, thus producing a mixture which after denaturation may give unexpected staining results. Pathological deposition of melanin occurs in a benign lesion called a nevus or ‘mole’. The malignant counterpart to the nevus is the malignant melanoma; it is in the diagnosis of this important tumor and its metastases that histological demonstration of melanin finds its most important practical application ( Fig. 14.5 ). Melanin is also found in the hair follicles of dark-haired people.

• 2.

  Eye , where it is found normally in the choroid, ciliary body and iris. Similar brown-black pigment is found in the retinal epithelium but its identity with melanin is uncertain. Melanomas can occur in the eye but these tumors are rare.

• 3.

  Brain , where it is found particularly in the substantia nigra in such quantities that this structure is macroscopically visible as a black streak on both sides of the mesencephalon. In patients with long-standing Parkinson’s disease this area is markedly reduced. Black melanin is also found in patches in the arachnoid covering some human brains, and has been described as having a ‘sooty’ appearance.

Demonstration of melanin

A number of methods can be used for the identification of melanin and melanin-producing cells. The most reliable of these are:

• 1.

  Reducing methods such as the Masson-Fontana silver technique and Schmorl’s ferric-ferricyanide reduction test.

• 2.

  Enzyme methods e.g. DOPA reaction.

• 3.

  Solubility and bleaching methods.

• 4.

  Fluorescent methods.

• 5.

  Immunohistochemistry.

Melanin and its precursors are capable of reducing both silver and acid ferricyanide solutions. It also shows the marked physical property of being completely insoluble in most organic solvents which is almost certainly due to the fact that formed melanin is tightly bound to protein within the melanosome. The other physical characteristic shown by melanin is its ability to be bleached by strong oxidizing agents. This property is particularly useful when trying to identify nuclear detail in heavily pigmented melanocytic tumors. Further reference to these procedures will follow the conventional demonstration techniques for melanin. These two physical characteristics relate to formed melanin and not to melanin precursors.

The enzyme tyrosinase can be demonstrated by the DOPA reaction and is demonstrable in any cell capable of synthesizing melanin. Cells which have produced an abundance of melanin, and in which the melanosomes are filled with pigment, are said to no longer show tyrosinase activity, but some workers have found that tyrosinase is active in most cells, even though melanin may be present in large quantities.

The fluorescent method depends upon the ability of certain biogenic amines, including DOPA and dopamine, to show fluorescence after exposure to formaldehyde (formalin-induced fluorescence). This method therefore demonstrates melanin precursors rather than formed melanin. Recent advances in antibody production have produced a wide range of antibodies which recognize antigens in the melanin synthesis pathway, e.g. tyrosinase or tyrosinase related protein 1 and 2 (TRP 1 and 2), or those recognizing melanocyte activation antigens, e.g. gp100 (HMB 45) and Mart-1 (Melan A). The use of enzyme histochemical procedures is now rarely required.

Reducing methods for melanin

Melanin is a powerful reducing agent and this property is used to demonstrate melanin in the following ways:

• 1.

  The reduction of ammoniacal silver solutions to form metallic silver without the use of an extraneous reducer is known as the argentaffin reaction. method (using Fontana’s silver solution) and its various modifications, which also rely on melanin’s argentaffin properties, are now widely used for routine purposes. Melanins are blackened by acid silver nitrate solutions. Melanin is also argyrophilic, meaning that melanin is colored black by silver impregnation methods which use an extraneous reducer ( Fig. 14.6 ). This is not a property considered to be of diagnostic value.

  

• 2.

  Melanin will reduce ferricyanide to ferrocyanide with the production of Prussian blue in the presence of ferric salts (the Schmorl reaction). This type of reaction ( Fig. 14.7 ) is also seen with certain other pigments e.g. some lipofuscins, bile and neuroendocrine cell granules.

  

• 3.

  Other reducing methods for demonstrating melanin are Lillie’s ferrous ion uptake (described in ) and Lillie’s Nile blue A ( ).