Pigmentation - Pathology

Exogenous Pigmentation

• Enter body from environment

Carbon (Anthracosis)

• This occurs where carbon particles are inhaled over a long period of time.
• Carbon particles are phagocytosed by alveolar Macrophages.
  • Are carried to a peribronchiolar location and the local bronchial lymph nodes.
• It is common in animals that live in cities.
  • There is air pollution due to smoke.
  • Mostly seen in dogs.
• Can occur in the mesenteric lymph nodes of pigs that feed on ash heaps.
• Carbon is insoluble and results in minimal irritation.

Gross Appearance

• Black or grey speckles in lungs.

Histological Apprearance

• Black extra- or intra-cellular granules .
  • Resistant to solvents and bleaching agents.
  • Can cause slight fibrosis in alveolar walls.

Pneumoconiosis

• A serious pigmentation.
• Results from repeated inhalation of irritating mineral dust, e.g.
  • Coal
  • Silica
  • Asbestos
• Leads to pulmonary fibrosis.
  • Varying shades of grey.
• Asbestos may also cause pulmonary, pleural and peritoneal tumours.
• Seen more in man than animals.
  • Occupational hazards.

Carotenoids

• Also known as lipochrome.
• Carotenoids are fat-soluble greenish-yellow pigments that bind to lipids in body.
  • Derived from carotenes and xanthophyll in plants.
  • Can be seen grossly in a number of normal places
    • Adrenal cortex.
    • Testes.
    • Corpus luteum.
    • Liver.
    • Yolks of eggs.
    • Fat of horses and Channel Island cattle.
• May be confused with a jaundiced liver or subcutis.
  • Adding minced tissue to a mixture of ether and water can distinguish the two.
    • Carotenoids are fat-soluble and dissolve in the ether fraction.
    • Bile pigments dissolve in the water fraction.

Iatrogenic

• E.g.
  • Iron injections in piglets.
  • Coloured antiseptics.
  • Tattooing.

Endogenous Pigmentation

• Pigments are formed inside the body.

Melanin

• Normally gives visible colour to the skin, hair, and iris.
• Melanin synthesis is controlled by melanocyte stimulating hormone (MSH).
  • MSH is produced in the pituitary.
  • Melanin is derived from tyrosine (an amino acid) utilising a copper-containing enzyme, tyrosinase.
• Melanosis occurs when excessive quantities of pigment are deposited in internal organs.
  • The lungs and aorta, pleura, brain and meninges, and liver are often affected.

Gross Appearance

• Black/brown pigmentation is seen, either as spots or as larger areas.
  • Larger areas can give the lungs or liver a chequer-board pattern.
• No change in structure occurs.

Histological Appearance

• Round granules of brown/black are seen in the cytoplasm of cells.
  • Can be bleached out by chlorine or KMNO₄.
  • Stain black with silver stain Masson Fontana.

Significance

• There is no significance of melanosis when it occurs in the internal organs.
• There is also no significance in hyperpigmentation of the skin.
• Melanosis may be associated with chronic irritation.
  • For example, in wound healing.
    • Scars are pigmented.
• Hyperpigmentation may be significant in excess cortisol states such as Cushing’s Disease.
  • There may be tumours of melanocytes.
• Albinos have no pigment in their hair or skin, and have pink irises.
  • Lack sufficient tyrosinase enzyme in their melanocytes.
  • Albino rabbits, rats and mice are seen.

Blood Pigments

• Blood pigments causing pigmentation may be
  • Normal derivatives of haemoglobin, which have accumulated in excess.
    • Haemosiderin.
    • Bilirubin.
  • Pathological
    • Methaemoglobin (chocolate brown) in nitrate poisoning.
    • Carboxyhaemoglobin (bright cherry red) in carbon monoxide poisoning.
    • Parasitic acid haematin.

Haemoglobin

• Results from excessive phagocytosis or haemolysis of red blood cells.
  • Haemoglobin is freed into the plasma which becomes red.
• If there is sufficient haemolysis, the haemoglobin overspills into the urine.
  • Haemoglobinuria.
    • Both the kidneys and the urine appear dark red.
• Can be caused by a variety of agents including:
  • Bacteria
    • E.g. Leptospira
  • Protozoa
    • E.g. Babesia bigemina
  • Chemicals
    • E.g. excessive copper in the liver of sheep.
  • Immune mediated conditions
    • E.g. autoimmune mediated haemolytic anaemia.
• Haemoglobin is not usually seen in the tissues.

Haemosiderin

• Haemosiderin is a golden brown/yellow pigment.
  • Derived from splitting haemoglobin into an iron-porphyrin compound and globulin.
    • This splitting occurs when ageing red blood cells are engulfed by macrophages in the spleen, lymph nodes, bone marrow and liver.
      • The iron containing fragment remains in the macrophages and is recycled to make more haemoglobin.
• Excessive quantities of haemosiderin arise from increased red cell breakdown by:
  • Phagocytosis
  • Haemolysis
• Large amounts of haemosiderin diffusely spread throughout the spleen usually indicates a haemolytic disorder.
• Local production of haemosiderin is responsible for the appearance of bruising in injury.
  • Other haemoglobin breakdown products are also formed.
• Haemosiderin also accumulates in chronic congestion of the vascular system due to heart failure.
  • Seen in highly vascular tissues such as the lung, liver and spleen.

Gross Appearance

• Grossly, very little can be seen unless there has been substantial breakdown of haemoglobin.
  • In this case the organ may appear brownish.
• Also seen as bruising.
• Usually not appreciable in the lung in heart failure.
• In repetitive exercise induced pulmonary haemorrhage of horses, the affected area of the dorsal lung may be brownish in colour.
• The equine liver often contains stores of haemosiderin giving them a brownish colour.

Histological Appearance

• Accumulates in the cytoplasm of macrophages.
• Chronic congestion in the lung can be seen due to heart failure.
  • Some of the red blood cells escape out of the alveolar capillaries and are engulfed by alveolar macrophages.
    • Called "heart failure" cells.
• Prussian blue stains specifically for haemosiderin.
  • Granules stain blue.

Haematin

• There are 2 types of haematin.
  • Acid haematin
  • Parasitic haematin.

Acid Haematin

• A pigment artefact.
• Acid in unbuffered formalin binds with haemoglobin to form a brownish-black granular pigment.
• Only seen microscopically.
• Can be formed in areas of ante-mortem ulceration of the stomach.
  • There is increased acidity due to hydrochloric acid.
• Acid haematin is iron negative.
• Must remove acid haematin prior to staining sections to allow haemosiderin to be distinguished.
  • A useful clue in the untreated section is the presence of the pigment in the red blood cells of vessels.

Parasitic Haematin

• Certain parasites in other parts of the world (e.g. Schistosoma sp.) take up blood.
  • Break haemoglobin to haematin.
  • Regurgitate the pigment back into circulation.
    • Taken up by macrophages, which go the lymph nodes, liver and spleen.
• Fasciola hepatica in this country causes chronic damage to the bile ducts of the bovine liver.
  • Produces acid haematin, which lines the bile ducts.
  • The fluke and its spines damage the duct lining and feed on blood liberated from the blood vessels.
    • This is regurgitated by the fluke and passes down the bile duct to the intestine together with the proteins liberated.
      • This parasitic haematin is in an un-resorbable form, and so the flukes lead to hypoproteinaemia and anaemia.

Jaundice

• Jaundice is also known as icterus.
• Jaundice is a yellowish discoloration due to excessive levels of bilirubin in plasma.
  • Seen in tissues that are normally white, e.g.
    • Fat
    • Intima of blood vessels
    • Sclera
• Bilirubin stains brown with H&E, like both haemosiderin and lipofuscin.
  • Must be distinguished from each other by special stains.
    • Bilirubin stains bright green with a Fouchet stain.

Production of Bilirubin

• Red blood cells either undergo:
  • Phagocytosis, in the case of ageing cells.
  • Haemolysis, in haemolytic crises.
• Haemoglobin is freed from the red cells and is further broken down in the reticulo-endothelial system to haeme and globulin.
  • Haeme is a mixture of iron and porphyrin.
  • Both the iron and globulin are recycled for further use in erythropoiesis.
• The porphyrin from haemoglobin breakdown is converted to biliverdin.
  • A green pigment
    • May contribute to the greenish appearance seen in local bruising.
• Biliverdin is subsequently changed into bilirubin.
• The bilirubin is then bound to albumin, and transported in the blood to the liver.
  • In the hepatocyte, bilirubin is released from the albumin and conjugated with glucuronic acid.
    • Forms conjugated bilirubin.
      • Excreted into the intestine via the bile ducts.

Prehepatic (Haemolytic) Icterus

• Due to excessive production of bilirubin due to haemolysis, e.g.
  • Babesiosis
  • Leptospirosis
  • Copper poisoning in sheep.
• There is excessive unconjugated bilirubin in the blood.

Hepatic (Toxic) Icterus

• Due to liver cell damage.
  • In hepatocytic necrosis, the liver cannot conjugate bilirubin.
    • Results in increased levels of unconjugated bilirubin in the blood.
  • In less severe damage, the hepatocytes swell and bile caniculi are pressed closed.
    • There is therefore intrahepatic obstruction conjugated bilirubin accumulated in the blood.
• In reality, these situations occur together.
  • There is both excessive unconjugated and conjugated bilirubin in the blood.
• Examples:
  • Leptospira icterohaemorrhagica in the dog.
  • Rift Valley fever virus.
  • Some plant poisonings.
    • Lantana.
  • Some fungal infections.
    • Aflatoxicosis.

Post-Hepatic (Obstructive) Icterus

• Due to the flow of bile being obstructed, for example by:
  • Gallstones
  • Parasites
    • Ascaris suum in the bile ducts of pigs.
  • Tumours
    • Adenocarcinoma of pancreas presses upon and occludes the bile duct.
• Results in excessive conjugated bilirubin in the blood.
• Constructive icterus causes, out of the three types of icterus,
  • The most elevated levels of bilirubin in the blood
  • The greatest discoloration of the tissues of the body.

Van de Berg Test

• This test can distinguish between the different types of jaundice, based on whether the bilirubin is conjugated or not.
• Plasma from the jaundiced animal is treated with an aqueous solution of a reagent (diazotised sulphanilic acid).
  • Gives a red-purple colour.
    • The intensity of this colour is directly proportional to the amount of water-soluble (i.e. conjugated ) bilirubin in the sample.
  • Further addition of alcohol intensifies the colour if there is non-water-soluble (i.e. unconjugated) bilirubin also present.
    • The intensified colour is directly proportional to the total amount of bilirubin present in the sample.
  • The difference between the two readings gives the amount of unconjugated bilirubin in the sample.

Haematoidin

• Haematoidin is bile pigment laid down in areas of focal haemorrhage where red blood cells are lysed.
  • I.e. it is unconjugated bilirubin in tissues.
• Has a clear yellow colour.
• Can sometimes be granular.
  • Could be confused with haemosiderin.
    • Unlike haemosiderin, haematoid does not contain iron.
      • Does not stain with Prussian Blue.

Porphyria

• Porphyrin is a component of haeme.
• Porphyria is a rare heritable condition in cattle, pigs and cats, where there is imperfect synthesis of porphyrin.
  • Due to defective enzymes.
  • Leads to a degree of anaemia in affected animals.
• Porphyria is best-documented in cattle.
  • Porphyrins (brownish in colour) accumulate in bone, teeth and other internal tissues.
  • Excess porphyrins also circulate in the blood stream and are excreted in the urine.
  • Urine has a reddish colour.
• Porphyrin is a fluorescent pigment.
  • Urine will also fluoresce under ultra-violet light.

Histological Appearance

• Brown granular pigment that looks like haemosiderin but is iron negative.
• Found in macrophages of bone marrow, liver, lungs, and lymph nodes.

Photosensitisation

• Porphyrin is a fluorescent pigment.
  • Is related to chlorophyll.
• UV light acts on porphyrins as they circulate in the blood under the white skin.
  • Changed to a longer wavelength.
    • Causes acute inflammation in the skin.
• The inflammation caused may look like sunburn, but is actually a necrotising dermatitis.
  • Affected areas slough off.
  • Large areas of the dermis are exposed to infection.
  • Death due to toxaemia can ensue if it is severe.
• Removing animals from sunlight causes the lesions to regress.

• The most common and important form of photosensitisation in animals is due to liver damage coupled with a diet containing a lot of chlorophyll.
  • I. e. animals on green pasture.
• A wide variety of plants and fungi have been implicated in causing acute toxic damage to the liver.
  • Including grasses, clovers and Brassica sp.
  • This liver damage may cause the imperfect breakdown of chlorophyll and raised phyloerythrin levels.
    • Causes photosensitisation.
• The common form in sheep is facial eczema.
  • The face and head are swollen, raw and bleeding.

• A third form of photosensitisation occurs in the absence of liver damage and is due to certain plants containing excessive fluorescent pigments, e.g.
  • Buckwheat (Fagopyrum esculentum)
  • St. John’s wort (Hypericum perforatum)

Lipofuscin

• A yellow-brown granular pigment composed of lipid, phospholipid and protein polymers.
  • Thought to be derived from the lipid peroxidation of cellular membranes.
• Lipofuscin is the ageing pigment.
  • Can be seen in senile animals in
    • Muscle
      • E.g. in "brown atrophy".
    • Myocardium
    • Neurones (particularly those of horses)
    • Normal liver of middle-aged cats.
• Xanthosis is when lipofuscin is a prominent feature in the tissues of Ayrshire cattle.
  • Lipofuscin is found in the heart, diaphragm, masseter muscles, kidney and adrenal cortex.
• Pathologically, lipofuscin is associated with Vitamin E/ Selenium deficiency.
  • There is excessive lipid peroxidation of cellular membranes by free O₂ radicals.

Gross Appearance

• Xanthosis - gives a brown colour to the organ.
• Senility
  • May impart a brownish colour to the heart.
    • Sometimes called ‘brown atrophy’.
  • In other areas, lipofuscin is only visualised microscopically.

Histological Appearance

• Granular
• Brown- yellow in colour.
• Lies in the cytoplasm...
  • ...At either end of the nucleus in the heart.
  • ...Scattered throughout in the liver and neurones

Ceroid

• Is a variant of lipfuscin.
• Stains acid fast.
• Occurs in:
  • Hepatocytes
    • E.g. in choline deficiency in cirrhosis
  • Macrophages
  • A variety of tissues including fat, cardiac muscle, splenic trabeculae and intestine in Vitamin E deficiency.
    • Sometimes being so prominent as to be grossly visible.
      • Yellow fat disease
        • Pigs and cats
      • Brown gut
        • Dogs