1 Endogenous pigments

These substances are produced either within tissues and serve a physiological function, or are by-products of normal metabolic processes. They can be further subdivided into:

2 Artifact pigments

These are deposits of artifactually produced material caused by the interactions between certain tissue components and some chemical substances, such as the fixative formalin. Formalin and malaria pigments are sometimes classified as a subdivision of endogenous pigments.

3 Exogenous pigments and minerals

These substances gain access to the body accidentally through a variety of methods (e.g. carbon anthracotic pigment, which is seen in lung and lymph nodes). These pigments and minerals serve no physiological function. Entry is gained either by inhalation into the lungs or by implantation (e.g. tattoos) into the skin. Most exogenous pigments are minerals, few of which are pigmented.

The above classification, although not scientifically precise, is used as a convenient aid in the identification of pigments. It is important to bear in mind that the same pigment may present itself in tissue sections in a variety of ways. Iron, for example, may be present as an endogenous pigment in liver sections in an iron overload condition and as an exogenous pigment in the case of a shrapnel wound/prosthetic implantation. It is advisable to note a pigment’s morphology, tissue site, and relevant clinical data before carrying out the various stains and histochemical reactions available in order that a pigment can be identified.

Hemosiderins

These pigments are seen as yellow to brown granules and normally appear intracellularly. They contain iron in the form of ferric hydroxide that is bound to a protein framework and is unmasked by various chemicals. Iron is a vital component of the human body as it is an essential constituent of the oxygen-carrying hemoglobin found in the red cells, where 60% of the body’s total iron content resides. It also occurs in myoglobin and certain enzymes, such as cytochrome oxidase and the peroxidases.

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 also the main site of iron utilization in the body, 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 that 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, 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, because 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 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, 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 that 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. 13.1). 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 that iron appears in its reduced state as the ferrous salt, then Lillie’s (1965) method may be used to achieve the Turnbull’s blue reaction to visualize its presence in tissue sections (Fig. 13.2).

Figure 13.1 A section of liver from a patient with hemochromatosis stained for ferric iron with Perls’ method.

Ferric iron is stained blue.

Figure 13.2 A section of placenta treated with ferrous sulfate and stained with Lillie’s method for ferrous iron.

Ferrous iron is stained dark blue.

An interesting and sometimes useful modification of a serum iron technique was introduced by Hukill and Putt (1962) 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.

Hemoglobin

Hemoglobin is a basic conjugated protein that is responsible for the transportation of oxygen and carbon dioxide within the bloodstream. It is composed of a colorless protein, globin, and a red pigmented component, heme. Four molecules of heme are attached to each molecule of globin.

Heme is composed of protoporphyrin, a substance built up from pyrrole rings and combined with ferrous iron. Histochemical demonstration of the ferrous iron is possible only if the close binding in the heme molecules is cleaved. This can be achieved by treatment with hydrogen peroxide, but this has no practical use. As hemoglobin appears normally within red blood cells its histological demonstration is not usually necessary. The need to demonstrate the pigment may arise in certain pathological conditions such as casts in the lumen of renal tubules in cases of hemoglobinuria or active glomerulonephritis.

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. Lison (1938) introduced the patent blue method that was later modified by Dunn and Thompson (1946) (Fig. 13.3). Tinctorial methods have also been used for the demonstration of hemoglobin; the amido black technique (Puchtler & Sweat 1962) and the kiton red-almond green technique (Lendrum 1949) are worth noting.

Figure 13.3 A section of kidney from a patient with hemoglobinuria stained for hemoglobin with leuco patent blue V.

Hemoglobin is stained blue.

Leuco patent blue V method for hemoglobin (Dunn & Thompson 1946)

Fixation

Formalin (removed formal mercury due to toxicity?) Poor preservation with Heidenhain’s Susa has been noted by Drury and Wallington (1980).

Solutions

Stock solution

1% aqueous patent blue V (CI 42045)	25 ml
Powdered zinc	2.5 g
Glacial acetic acid	0.5 ml

Mix well on a magnetic stirrer. The solution will become pale green-blue. Filter and store in a refrigerator at 3–6°C. The solution is stable for about 1 week.

Working solution

Stock solution	10 ml
Glacial acetic acid	2 ml
3% hydrogen peroxide	1 ml

Prepare immediately before use.

Method

1. Take test and control sections to distilled water.

2. Stain in patent blue solution for 5 minutes at room temperature.

3. Rinse in distilled water.

4. Lightly counterstain in 0.5% aqueous neutral red or 0.1% aqueous nuclear fast red for 1 minute.

5. Rinse in distilled water.

6. Dehydrate, clear, and mount in synthetic resin.

Results

Hemoglobin peroxidase (red blood cells and neutrophils)	dark blue
Nuclei	red

Notes

a. Experience has shown that the hemoglobin demonstrated by this method tends to be more of a green-blue color.

b. In some instances, if the staining solution is left on the section for too long, it may start to decolor a positive reaction.

c. Fixation in excess of 36 hours may give rise to unreliable results.

d. This method demonstrates peroxidase activity, including the peroxidases in other blood cells, particularly in the lysosomes of polymorphonuclear leukocytes and their precursors. Tissue peroxidases are also demonstrated.

Bile pigments

Red blood cells are broken down in the reticuloendothelial system when they have reached the end of their useful life, usually after 120 days. Hemoglobin is released after the red cell membrane has been ruptured. The protein globin and iron components are released for recycling within the body after the hemoglobin has been broken down. After the heme portion has been split from the globin, the tetrapyrrole ring of the heme molecule is cleaved and opened out into a chain composed of four linked pyrrole groups. With the opening out of the tetrapyrrole ring, the iron component is removed to be stored in those tissues that specialize in iron storage. This iron component is now free to be incorporated into the hemoglobin molecule during red cell formation. The opened tetrapyrrole ring, which has had its iron component removed, is known as biliverdin. This residue is formed in the phagocytic cells that populate the reticulo-endothelial system, particularly the bone marrow and spleen. Biliverdin is transported to the liver, where it is reduced to form bilirubin. In this form, the bilirubin is insoluble in water, but after conjugation with glucuronic acid it forms a water-soluble compound, bilirubin-glucuronide. This process takes place in liver hepatocytes due to the activity of the enzyme glucuronyl transferase. The conjugated bilirubin passes from the hepatocytes into the bile canaliculi, and then via the hepatic ducts into the gallbladder, which acts as a reservoir. The bilirubin passes along the common bile duct to be released into the duodenum via the ampulla of Vater.

The term bile pigments used by many authors when discussing the various (generic) staining techniques can be applied to all bile pigments. In using this terminology, it is implied that all bile pigments react in an identical manner, but this is not the case. Contained within the group ‘bile pigments’ are both conjugated and unconjugated bilirubin, biliverdin, and hematoidin, all of which are chemically distinct and show different physical properties, particularly with regard to their solubility in water and alcohol. Microscopic examination of any liver section that contains ‘bile pigments’ will almost certainly reveal a mixture of biliverdin and both conjugated and unconjugated bilirubin. This is particularly likely when the liver contains an excess of bile pigments, either through bile duct obstruction, e.g., due to a stone or tumor, an abnormality of biliverdin-bilirubin metabolism in the rare congenital enzyme disorders, or where there is extensive liver cell death or degeneration. The non-specific term bile will be used to include biliverdin and both conjugated and unconjugated bilirubin in the following text.

In a hematoxylin and eosin (H&E) stained section of liver, bile, if present, is most commonly seen in the hepatocytes in the early stages as small yellow-brown globules and then subsequently within the bile canaliculi as larger, smooth, round-ended rods or globules commonly referred to as bile thrombi. The latter, if present, in liver sections is a histopathological indication that the patient has obstructive jaundice due to a blockage in the normal flow of bile from the liver into the gallbladder and subsequently into the bowel, probably because of gallstones or a carcinoma of the head of pancreas. Masses of bile in the canaliculi of liver sections are easily distinguished microscopically because of their characteristic morphology and their situation. Bile in hepatocytes must be distinguished from the lipofuscins that are also commonly seen within these cells and can appear as small yellow-brown globules. The need to distinguish between bile and lipofuscin in hepatocytes is particularly important in liver biopsies taken from liver transplant patients where sepsis is suspected. Bile is difficult to identify in the sections of normal liver. It is important to note that both bile and lipofuscin can be positive with Schmorl’s ferric ferricyanide reduction test (Golodetz & Unna 1909). Bile is also seen in H&E-stained sections in the gallbladder where it can appear as amorphous, yellow-brown masses adherent to the mucosa or included as yellow-brown globules within the epithelial-lined Aschoff-Rokitansky sinuses in the gallbladder. Bile is also present, together with cholesterol, in gallstones.

Virchow (1847) first described extracellular yellow-brown crystals and amorphous masses within old hemorrhagic areas, which he called hematoidin. Pearse (1985) reviewed the histochemistry of bile pigments. Microscopically, hematoidin frequently appears as a bright yellow pigment in old splenic infarcts, where it contrasts well against the pale gray of the infarcted tissue. Hematoidin can also be found in old hemorrhagic areas in the brain. Bearing in mind the differences discussed above, it is almost certain that hematoidin is related to both bilirubin and biliverdin, even though it differs from them both morphologically and chemically. It is thought that heme has undergone a chemical change within these areas which has led to it being trapped, thus preventing it from being transported to the liver to be processed into bilirubin.

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 sections, and it is worth remembering that the green color of biliverdin is often masked by eosin. In such cases, unstained paraffin or frozen sections, lightly counterstained with a suitable hematoxylin (e.g. Mayer), 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 (Hall 1960), 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. 13.4). The Fouchet technique is quick and simple to carry out, and when counterstained with van Gieson’s solution the green color is accentuated.

Figure 13.4 Bile in a section of liver stained with Hall’s method for bilirubin.

Bilirubin is stained emerald green.

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 that has been used to demonstrate bile pigments is the diazo methods that 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. Raia 1965, 1967) 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.

Porphyrin pigments

These substances normally occur in tissues in only small amounts. They are considered to be precursors of the heme portion of hemoglobin. The porphyrias are rare pathological conditions that are disorders of the biosynthesis of porphyrins and heme.

In erythropoietic protoporphyria, porphyrin pigment can be seen as focal deposits in liver sections. The pigment appears as a dense dark brown pigment and in fresh frozen sections exhibits a brilliant red fluorescence that rapidly fades with exposure to ultraviolet light. The pigment, when seen in paraffin sections and viewed using polarized light, appears bright red in color with a centrally located, dark Maltese cross.

Melanins

Melanins are a group of pigments whose color varies from light brown to black. The pigment is normally found in the skin, eye, substantia nigra of the brain, and hair follicles (a fuller account of these sites is given later.) Under pathological conditions, it is found in benign nevus cell tumors and malignant melanomas. The chemical structure of the melanins is complex and varies from one type to another. Melanin production is not fully understood but the generally accepted view is that melanins are produced from tyrosine by the action of an enzyme tyrosinase (syn. DOPA oxidase). This enzyme acts on the tyrosine slowly to produce the substance known as DOPA (dihydroxyphenylalanine) which is subsequently rapidly acted upon by the same enzyme to produce an intermediate pigment which then polymerizes to produce melanin. The later stages of melanogenesis remain largely speculative, and it is beyond the scope of this chapter to evaluate the many studies relating to melanin biosynthesis that have been carried out recently. Pearse (1985) gives a more detailed account of melanin production.

The melanins are bound to proteins, and these complexes are localized in the cytoplasm of cells within so-called ‘melanin granules’. Ghadially (1982) described these granules as the end stage of the development of the melanosome as seen at ultrastructural level.

There are four recognized stages of melanosome maturation:

Ultrastructurally the lamellar structures become increasingly difficult to see following melanin deposition. By the time the melanosome reaches stage 4 the lamellae structures are completely obscured (Fig. 13.5).

Figure 13.5 Transmission electron micrograph of a stage 3 melanosome, demonstrating the characteristic internal lamellae structure.

(Courtesy of Ms Tracey Deharo.)

The enzyme tyrosinase cannot be demonstrated in the mature granule.

The most common sites where melanin can be found are

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

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 therefore demonstrable in any cell capable of synthesizing melanin. Cells that have produced an abundance of melanin, and in which the melanosomes are filled with pigment, are said no longer to 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), 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 two ways:

Masson-Fontana method for melanin (Fontana 1912; Masson 1914)

Fixation

Formalin is best; chromate and mercuric chloride should be avoided.

Sections

Works on all types of section, although some adjustment may be necessary for resin sections.

Preparation of silver solution (after Fontana)

Place 20 ml of a 10% aqueous silver nitrate solution in a glass flask. Using a fine-pointed dropper pipette, add concentrated ammonia drop by drop, constantly agitating the flask until the formed precipitate almost dissolves. This titration is critical if the method is to work consistently well. The end point of the titration is seen when a faint opalescence is present, and is best viewed using reflected light against a black background. If too much ammonia is inadvertently added then the addition of a few drops of 10% silver nitrate will restore the opalescence. To this correctly titrated solution add 20 ml triple distilled water and then filter into a dark bottle. Store the solution in the refrigerator and use within 4 weeks.

Note: Ammoniacal silver solutions are potentially explosive if stored incorrectly.

Method

1. Take test and control sections to distilled water.

2. Treat with the ammoniacal silver solution in a Coplin jar that has been covered with aluminum foil, for 30–40 minutes at 56°C or overnight at room temperature.

3. Wash well in several changes of distilled water.

4. Treat sections with 5% aqueous sodium thiosulfate (also known as hypo) for 1 minute.

5. Wash well in running tap water for 2–3 minutes.

6. Lightly counterstain in 0.5% aqueous neutral red or 0.1% aqueous nuclear fast red for 5 minutes.

7. Rinse in distilled water.

8. Dehydrate, clear, and mount in a synthetic resin.

Results

Melanin, argentaffin, chromaffin and some lipofuscins	black
Nuclei	red

Notes

a. Only use thoroughly clean glassware, as the silver solution will react with any residual contaminant left on glassware.

b. Prolonged exposure to 56°C may give rise to a fine deposit over the section.

c. Friable material may need to be coated with celloidin as the ammonia in the silver solution could lead to sections lifting off the slide.

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