Histochemistry of Pigment and minerals.pdf

Histochemistry:Pigment and minerals  pigments are defined as substances occurring in living matter that absorb visible light  Therefore, various pigments may greatly differ in origin, chemical constitution, and biological significance.  They can be either organic or inog...

Histochemistry:Pigment and minerals  pigments are defined as substances occurring in living matter that absorb visible light  Therefore, various pigments may greatly differ in origin, chemical constitution, and biological significance.  They can be either organic or inoganic compounds. Classification  Endogenous pigments  Artifact pigments  Exogenous pigments and minerals  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: a. hematogenous (blood-derived) pigments b. non-hematogenous pigments c. endogenous minerals. A Hematogenous This group contains the following blood-derived pigments: hemosiderins hemoglobin bile pigments porphyrins.
 Hemosiderins Hemosiderin are seen as golden yellow to brown, granules or crystalline pigment normally appear intracellularly. They contain iron in the form of ferric hydroxide that is bound to a protein framework apoferritin to form ferritin micelles. 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. Hemosiderin pigment represents aggregates of ferritin Significance Systemic overload of iron - hemosiderosis. The main causes of hemosiderosis are 1. increased absorption of diaetry iron due to an inborn error of metabolism called hemochromatosis. 2. hemolytic anemias, 3. Repeated blood transfusions. Localised Hemosiderin Deposits  Pulmonary hemosiderosis: seen in mitral stenosis and left ventricular heart failure cells. Demonstration of hemosiderin and iron Detection of ferric iron in tissues. Strongly bound iron as in hemoglobin will not react.  Methods of dermonstration Perls' Prussian blue reaction (Perls 1867) Hukill and Putt's method for ferrous and ferric iron (Hukill & Putt 1962) The method is very similar to Perls' Prussian blue, but uses ferricyanide instead of ferrocyanide. Lillie’s method for ferric and ferrous iron.  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.  Histochemical demonstration of the ferrous iron is possible only if the close binding in the heme molecules is cleaved. Demonstration of hemoglobin Two types of demonstration method can be used to stain hemoglobin in tissue sections. The first demonstrates the enzyme, hemoglobin peroxidase. This peroxidase was 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). Tinctorial methods have also been used for demonstration of hemoglobin; the Amido black technique (Puchtler & Sweat 1962) and the Kiton red-Almond green technique (Lendrum 1949).  Bile pigments RBCs 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 RBCs membrane has been ruptured. The protein, globin, and iron components are released for recycling within the body after the hemoglobin has been broken down. The heme portion is split from the globin. The iron component is removed and 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 tetra-pyrrole ring of the heme molecule is cleaved -and opened out into a chain composed of four linked pyrrole groups.  The opened tetra-pyrrole ring, which has had its iron component removed, is known as biliverdin. Biliverdin is transported to the liver where it is reduced to form bilirubin. 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.  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.  The green color of biliverdin is often masked by eosin.  Bile pigments are not auto fluorescent 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 trichloracetic acid.  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, shows as bright red in color with a centrally located, dark Maltese cross.  Non-hematogenous endogenous pigments  This group contains the following:  melanins  lipofuscins  chromaffin  pseudomelanosis (melanosis coli)  Dubin-Johnson pigment  ceroid-type lipofuscins  Hamazaki-Weisenberg bodies  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  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  The melanins are bound to proteins, and these complexes are localized in the cytoplasm of cells called 'melanin granules'.  Other methods for melanin demonstration Microwave ammoniacal silver method for argentaffin and melanin (Churukian 2005)  Masson-Fontana method for melanin (Fontana 1912; Masson 1914)  Schmorl's reaction (taken from Lillie 1954)  Formaldehyde-induced fluorescence method for melanin precursor cells (Eranko 1955)  Ferrous ion uptake reaction for melanin (Lillie & Fullmer 1976)  Nile blue method for melanin and lipofuscin (Lillie 1956)  Lipofuscins  These yellow-brown to reddish-brown pigments occur widely throughout the body and are thought to be produced by an oxidation process of lipids and lipoproteins.  The oxidation process occurs slowly and progressively, and therefore the pigments exhibit variable staining reactions, different colors, and variation in shape and size, which appears to be dependent upon their situation.  This type of pigment is found in the following sites:  Hepatocytes.  Cardiac muscle cells, particularly around the nucleus. Large amounts of pigment are found in the small brown hearts of elderly debilitated people, a condition known as 'brown atrophy of the heart'. Inner reticular layer of the normal adrenal cortex, where the pigment imparts a brown color, is particularly prominent in patients dying after a long and stressful illness.  Testis, particularly in the interstitial cells of Leydig; it is responsible for giving testicular tissue its brown color.  Ovary.  Cytoplasmic inclusions in the neurons of the brain, spinal cord, and ganglia.  Other tissues such as bone marrow, involuntary muscle, cervix, and kidney. Demonstration of lipofuscins Lipofuscin is formed by a slow progressive oxidation process of lipids and lipoproteins, histochemical reactions will vary according to the degree of oxidation present in the pigment when the demonstration techniques are applied. Therefore, it is advisable to carry out a variety of techniques in order to be sure whether the pigment is lipofuscin.  The lipofuscins react with a variety of histochemical and tinctorial staining methods, the most common and useful being:  Periodic acid-Schiff method  Schmorl's ferric-ferricyanide reduction test.  Long Ziehl-Neelsen method  Sudan black B method Gomori's aldehyde fuchsin technique  Masson-Fontana silver method  Basophilia, using methyl green  Churukian's silver method Lillie's Nile blue sulfate method  Chromaffin This pigment is normally found in the cells of the adrenal medulla as dark brown, granular material. It may occur in tumors of the adrenal medulla (pheochromocytomas). Fixation in formalin is not recommended, and fixatives containing alcohol, mercury bichloride, or acetic acid should be avoided. Orth's or other dichromate containing fixatives are recommended. Chromaffin may be demonstrated by Schmorl's reaction, Lillie's Nile blue A, the Masson-Fontana, Churukian's microwave ammoniacal silver method, and the periodic acid-Schiff (PAS).  Dubin-Johnson pigment  This pigment is found in the liver of patients with Dubin Johnson syndrome and is due to defective canalicular transport of bilirubin.  It is characterized by the presence of a brownish-black, granular, intracellular pigment situated in the centrilobular hepatocytes.  The true nature of the pigment is yet to be established, but histochemically, it is similar to lipofuscin, though there are ultrastructural differences  Ceroid-type lipofuscins  Lillie et al (1941,1942) were the first to describe ceroid in the cirrhotic livers of animals maintained on inadequate diets.  Lillie thought that ceroid was different from lipofuscin because it failed to stain with the ferric-ferricyanide reaction.  Pearse (1985) states that ceroid is infact a lipofuscin at an early stage of oxidation. Further oxidation would produce lipofuscin proper.  Hamazaki- Weisenberg bodies  These small, yellow-brown spindle-shaped structures are found mainly in the sinuses of lymph nodes, either lying free or as cytoplasmic inclusions, and their significance is unknown.  First described by Hamazaki (1938). they have been described as being present in lymph nodes from patients with sarcoidosis (Weisenberg 1966). Hall and Eusebi (1978) have reported their presence in association with melanosis coli.  Histochemically they are similar to lipofuscin, and at ultrastructural level have an appearance that suggests that they are probably giant lysosomal residual bodies (Doyle et aI1973).  ENDOGENOUS MINERALS Calcium Insoluble inorganic calcium salts are a normal constituent of bones and teeth. The free ionic form of calcium, found in the blood, cannot be demonstrated. Abnormal depositions of calcium can be found in necrotic areas of tissue associated with tuberculosis, infarction (Gandy-Gamna bodies), atheroma in blood vessels, and malakoplakia of the bladder (Michaelis-Gutman bodies). Calcium salts are usually monorefringent but calcium oxalate is birefringent. Calcium usually stains purple blue with H&E  Copper Many enzymes in the body would fail to function without the presence of copper, although copper deficiency is extremely rare. Copper accumulation is associated with Wilson's disease. the most important disorder of copper metabolism. This disease is a rare inherited autosomal recessive condition which gives rise to copper deposition in the liver, basal ganglia of the brain, and eyes. In the eye, the Kayser-Fleischer ring. a brown ring of deposited copper may be seen in the cornea (Descemet's membrane). Copper deposition in the liver is also associated with primary biliary cirrhosis and certain other hepatic disorders.  Uric acid and urates Uric acid is a break down product of the body's purine (nucleic acid) metabolism, but a small proportion is obtained from the diet. Most, but not all, uric acid is excreted by the kidneys. The uric acid circulating in the blood is in the form of monosodium urate, which in patients with gout may be high, forming a supersaturated solution. High levels of Uric acid may result in urate depositions, which are water soluble in tissues, causing:  subcutaneous nodular deposits of urate crystals ('tophi')  synovitis and arthritis  renal disease and calculi.  ARTIFACT PIGMENTS This group of pigments comprises:  formalin  malaria  schistosome  mercury  chromic oxide  starch.  Formalin pigment This pigment is seen as a brown or brownish-black deposit in tissues that have been fixed in acidic formalin. The deposit is usually present in blood-rich tissues such as spleen, hemorrhagic lesions, and large blood vessels filled with blood. The morphology of the pigment can vary but is commonly seen as a microcrystalline deposit that is anisotropic (birefringent). One way of removing this pigment from tissue sections is by treating unstained tissue sections with saturated alcoholic picric acid. Alcoholic solutions of both sodium and potassium hydroxide will also remove the pigment but these may have deleterious effects on subsequent staining techniques. The use of buffered neutral formalin will help to minimize the problem of formalin pigment deposition. Fixation of large blood-rich organs, such as spleen, for a long period will tend to increase the amount of formalin pigment formed. Under these conditions it is advisable to change the fixative on a  Malaria pigment This pigment is morphologically similar to formalin pigment and occasionally may be identical, even though it is produced in a slightly different manner. It is formed within, or in the region of, red blood cells that contain the malaria parasite. In cases of cerebral malaria, malaria pigment can be seen in the red blood cells within the tiny blood capillaries of the brain. The pigment may, on occasion, be so heavily deposited that it obscures the visualization of the malaria parasite. Malaria pigment may also be present within phagocytic cells that have ingested infected red cells, therefore one should carefully examine the Kupffer cells of the liver, the sinus lining cells of lymph nodes and spleen, and within phagocytic cells in the bone marrow. Malaria pigment, like formalin pigment, exhibits birefringence and can be removed from tissue sections with saturated alcoholic picric acid.  Schistosome pigment This pigment is occasionally seen in tissue sections where infestation with Schistosoma can be seen; the pigment, which tends to be chunky, shows similar properties to those of both formalin and malaria pigments. Mercury pigment This pigment is seen in tissues that have been fixed in mercury-containing fixatives. Mercury pigment varies in its appearance but it is usually seen as a brownish-black, extracellular crystal. Although usually seen as monorefringent, occasionally it is birefringent, particularly when formalin-fixed tissue has been secondarily fixed in formal mercury. A prolonged storage of stained sections that contain mercury pigment can bring about a change in the structure of the pigment. The pigment changes from a crystalline form to a globular one. The reason for this is unclear but it may be caused by interaction between the pigment and the mounting medium. Furthermore, the globular form exhibits a Maltese cross birefringence.  Chromic oxide This pigment is rarely seen in tissue sections. When seen, it presents as a fine yellow-brown particulate deposit in tissues, as a result of not washing in water, tissues that have been fixed in chromic acid or dichromate containing fixatives. Subsequent treatment of tissues with graded alcohols, as used in tissue processors, may result in the reduction of chrome salts to the chromic oxides, which are insoluble in alcohol. The pigment is monorefringent and extracellular. It can be removed from sections by treatment with 1% acid alcohol. Starch This pigment is introduced by talcum powder from the gloves of surgeons, nurses, or pathologists. It is PAS and Gomori methenamine silver (GMS) positive and can be easily identified by its characteristic appearance and because when polarized it will  EXOGENOUS PIGMENTS AND MINERALS This types of mineral gain access to the body by inhalation, ingestion, or skin implantation, as a result of industrial exposure. Occasionally mineral deposition may occur due to medication or wound dressing. Tattoo pigment This is associated with skin and any adjacent lymphoid areas. If viewed using reflected light, the various colors of the dye pigments used to create the tattoo can be seen. Carbon This exogenous substance is the most commonly seen mineral in tissues and is easily recognized in stained tissue sections. Commonly found in the lung of urban dwellers and tobacco smokers. the main sources of this mineral are car exhausts and smoke from domestic and industrial chimneys. Black pigmentation of the lung (anthracosis) is seen as a result of massive deposition of carbon in coal workers and some city dwellers.  Silica Silica in the form of silicates is associated with the majority of all mined ores because they are found in or near rocks that contain silica. Mine workers inhale large quantities of silica that can give rise to the disease silicosis. This disease presents as a progressive pulmonary fibrotic condition which gives rise to impaired lung capacity and in some cases extreme disability. Silicates are also abundant in stone and sand, and any industrial worker involved in grinding stone or sand blasting will be at risk from silicosis. Silica is unreactive, thus is not demonstrated by histological stains and histochemical methods. It is examined using polarized light.  Lead Environmental pollution due to lead has been greatly reduced in recent times. Lead pipes that carried much of the domestic water supply have been replaced by alternative materials. Lead in paint, batteries, and gasoline has been reduced by the various manufacturers. Cases of lead poisoning are rare and are usually diagnosed biochemically using the serum from suspected cases. In chronic lead poisoning, excessive amounts can be deposited within many tissues, particularly bone and kidney tubules For many years various methods have been used to demonstrate lead in tissue sections; the most popular method is the rhodizonate method (Lillie 1954). Other methods for lead include the sulfidesilver of Timm (1958) and the unripened  Beryllium and aluminum The same methods are used to demonstrate both of these metals. It is therefore convenient to consider both together. Beryllium gains access to the body by inhalation or traumatization of the skin. A foreign body granuloma is formed. Conchoidal (shell-like) bodies can also be found, which are typical of, but not specific to, beryllium. These bodies usually give a positive reaction with Perls' Prussian blue. Aluminum is rarely seen in tissues but gains access to the body in a similar way to beryllium. It can also be found in bone biopsies from patients with encephalopathy on regular hemodialysis for chronic renal failure. Prolonged dialysis can cause osteodystrophy. It can develop insidiously and may present with a non-specific ache. The most severe pain occurs with osteomalacia particularly when it is associated with aluminum deposition (Brenner 2004). Beryllium and aluminum can both be demonstrated by solochrome azurine that forms a deep blue chelate. Aluminum is also positive with the fluorescent Morin method (Pearse 1985). Naphthochrome green can also be used to  Silver Silver is rarely found in the skin of silver workers as a result of industrial exposure. It is now more commonly seen as a localized change in the mouth of amalgam tattoo, or in association with silver earrings in ineptly pierced lobes. The resultant permanent blue gray pigmentation is called argyria and is most marked in those areas exposed to sunlight. In unstained and H&E-stained sections the silver appears as fine dark brown or black granules, particularly in basement membranes and sweat glands. The method of Okamoto and Utamura (1938), a metal chelating method that utilizes dimethylaminobenzylidene-rhodanine, will demonstrate silver.