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PIGMENT METABOLISM PRESENTER - Dr SHREYA PRABHU MODERATOR - Dr ANISHA T S 1

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  • PIGMENT METABOLISM

    PRESENTER - Dr SHREYA PRABHU

    MODERATOR - Dr ANISHA T S

    1

  • INTRODUCTION

    PIGMENTS are colored substances, some of which are normal

    constituents of cell, whereas others are abnormal and accumulate

    in cells only under special circumstances.

    They absorb visible light within a narrow band between 400-800

    nm.

    Thus pigments greatly differ in origin, chemical constitution, and

    biological significance.

    They can be organic or inorganic compounds that remain insoluble

    in most solvents

    2

  • CLASSIFICATION

    A)ENDOGENOUS PIGMENTS

    1) HEMATOGENOUS PIGMENTS

    a. Hemosiderin

    b. Hemoglobin

    c. Bilirubin

    d. Porphyrins

    2) NON HEMATOGENOUS PIGMENTS

    a. Melanin

    b. Lipofuscins

    c. Chromaffin

    d. Pseudomelanosis

    e. Dubin-Johnson pigment

    f. Ceroid-type lipofuscins

    g. Hamazaki-Weisenberg bodies

    3

  • B)EXOGENOUS PIGMENTS

    Inhaled pigments

    Ingested pigments

    Injected pigments

    C)ARTIFACT PIGMENTS

    Formalin

    Malaria

    Schistosome

    Mercury

    Chromic oxide

    Starch

    4

  • ENDOGENOUS

    PIGMENTS

    5

  • HEMOSIDERINS

    Hemoglobin derived, GOLDEN YELLOW to BROWN granular intracellular

    pigments.

    They contain iron in the form of ferric hydroxide that is bound to a protein

    framework

    Formed by aggregates of ferritin (iron complexed to apoferritin) found

    especially within the phagocytes of the bone marrow, spleen, liver where the

    break down of senescent RBC takes place.

    Excessive storage of hemosiderin(hemosiderosis) occurs in situation where

    there is excessive breakdown of red cells or systemic overload of iron

    6

  • 7

    HEMOSIDEROSIS

    LOCALISED GENERALISED

    LOCAL TISSUES PARENCHYMAL DEPOSISTS

    (Macrophages, fibroblasts, endothelial (Liver, Kidney, Pancreas. Heart, Skin)

    cells and alveolar cells) RED CELL DEPOSISTS

    (Liver, Spleen, Bone marrow)

    Examples: Examples:

    1.Hemorrhage in tissues 1.Acquired Hemosiderosis

    2.Black eye 2.Hereditary Hemosiderosis

    3.Brown induration lung 3.Excessive dietary intake (Bantus

    4.Infraction disease)

  • 8

  • DEMONSTRATION OF HEMOSIDERIN AND

    IRON

    PERLS PRUSSIAN BLUE REACTION FOR FERRIC IRON:

    Considered to be first classical histochemical reaction.

    Treatment with an acid ferrocyanide solution will result in the unmasking of

    ferric iron in the form of the hydroxide, Fe(OH)3, by dilute hydrochloric acid.

    The ferric iron reacts with a dilute ferrocyanide solution to produce an insoluble

    blue compound, ferric ferrocyanide (prussian blue)

    FIXATION:

    Avoid the use of acid fixatives. Chromates will also interfere with the

    preservation of iron

    9

  • 10SECTIONS:

    Works well on all types of section, including resin

    FERROCYANIDE SOLUTION:

    1% aqueous potassium ferrocyanide 20 ml

    2% aqueous hydrochloric acid 20 ml

    Freshly prepared just before use

    METHOD:

    Take a test and control section to water

    Treat sections with the freshly prepared acid ferrocyanide solution for 10-30 minutes

    Wash well in distilled water

    Lightly stain the nuclei with 0.5% aqueous neutral red or 0.1% nuclear fast red

    Wash rapidly in distilled water

    Dehydrate, clear, and mount in synthetic resin

    RESULTS:

    Ferric iron Blue

    Nuclei Red

  • 11

    A SECTION OF LIVER FROM A PATIENT WITH HEMOCHROMATOSIS

    STAINED FOR FERRIC IRON WITH PERLSMETHOD. FERRIC IRON IS

    STAINED BLUE

  • 12

  • LILLIES METHOD FOR FERRIC AND FERROUS IRON

    Ferric iron dark Prussian blue

    Ferrous iron dark Turnbulls blue

    Nuclei Red

    HUKILL AND PUTTS METHOD FOR FERROUS AND

    FERRIC IRON

    Ferrous iron Red

    Nuclei Blue

    13

  • 15

    A SECTION OF PLACENTA TREATED WITH LILLIES METHOD FOR

    FERROUS IRON. FERROUS IRON IS STAINED DARK BLUE

  • HEMOGLOBIN

    HEMOGLOBIN is a basic conjugated protein bound to globin and is the red

    pigment component, responsible for the transportation of oxygen and carbon

    dioxide.

    Heme is composed of protoporphyrin, a substance built up from pyrrole rings

    and combined with ferrous iron.

    Histochemical demonstration of the ferrous iron is only possible if the close

    binding in the heme molecules is cleaved

    17

  • As Hb is normally present within red blood cells its

    demonstration is not necessary.

    Outside its normal position in RBC, Hb may be found free in

    areas of recent hemorrhage, in macrophages.

    The pathological conditions like casts in the lumen of renal

    tubules in cases of hemoglobinuria or active

    glomerulonephritis.

    18

  • DEMONSTRATION OF HEMOGLOBIN

    Methods demonstrate the enzyme, Hemoglobin peroxidase,

    which is reasonably stable and withstands short fixation and

    paraffin processing.

    This peroxidase activity was demonstrated by the Benzidine-

    nitroprusside methods ( Lepehne-Pickworth Benzidine

    Trchnique), but because of the carcinogenicity of benzidine, these

    methods are not recommended.

    Tinctorial method, The amido black technique and the Kiton

    red-Almond green technique are worth noting

    19

  • 20

    LEUCO PATENT BLUE METHOD

    Hemoglobin peroxidase Dark Blue

    Nuclei Red

  • BILE PIGMENTS

    23

  • 24Heme Biliverdine Bilirubin(unconjugated)

    Bilirubinalbumin complex

    (Uptake by liver)

    Conjugated

    bilirubin Bilirubin-diglucuronide

    in intestine Urobilinogen

    StercobilinogenUrobilinogen

    In kidney

    Urobilin

    Excretion in urine

    Stercobilin

    Excretion in feces

    20% absorptionEnterohepatic

    circulation

    80% Intestine

    oxygenase

    Bilirubin

    reductase

    Heme

    Glucuronyl

    transfersae

  • BILE PIGMENTS

    Bilirubin (conjugated+unconjugated), biliverdine, hematoidin-

    together refered to as Bile pigments

    They are chemically and physically distinct with solubility in

    water and alcohol

    Bilirubin is the orange-yellow pigment, a toxic waste product

    in the body.

    It is extracted and biotransformed mainly in the liver, and

    excreted in bile and urine.

    25

  • HEMATOIDIN-

    Virchow first described in sites of old hemorrhage

    Related to bile pigments but differ

    Thought that heme has undergone a chemical change within these areas- led to it being trapped- preventing transportation to liver

    Extracellular yellow-brown crystals and amorphous masses within old hemorrhagic areas

    Microscopically- appear as bright yellow pigment in sections of old splenic infarcts, old hemorrhagic areas of brain or infarcted tissues

    26

  • Microscopical examination of any liver sections that contains bile

    pigments will almost certainly reveal a mixture of biliverdine and

    both conjugated and unconjugated bilirubin

    In H&E stained sections- bile if present-

    seen as small yellow brown globules within bile canaliculi- indicating

    obstruction

    Within hepatocytes (they need to be distinguished from Lipofuscin)

    Conditions- Prehepatic/ Hepatic/ Post hepatic

    27

  • 28

  • 29

    Intracellular

    Cholestasis,

    Bile pigments in

    The cytoplasm

    Fig: CHOLESTASIS

  • 30

    BILE PLUG (arrow) showing expansion of bile canaliculus by bile

  • DEMONSTRATION OF BILE PIGMENTS:

    Need arises in the histological examination of liver where

    distinguishing from lipofuscin is of significance

    Both appear yellow-brown in H&E paraffin sections

    Bile pigments are not autofluorescent and fail to rotate the

    plane of polarized light, whereas Lipofuscin is autofluorescent

    Most common method- Modified Fouchet Technique

    31

  • MODIFIED FOUCHETS (HALL) TECHNIQUE

    (FOR LIVER BILE PIGMENTS)

    32

    RESULTS-

    Bile pigments emerald to blue green

    Muscle yellow

    Collagen red

  • 35

  • OTHER TECHNIQUES

    GMELIN TECHNIQUE-

    Only method that shows identical result with liver, gallbladder bile and

    hematoidin.

    Method- Deparaffinized sections of tissue treated with nitric acid and

    changing color spectrum is produced around pigment deposits

    Red Purple Green

    KUTLIKS TECHNIQUE-

    Method-Sections treated with ferric iron solution

    Result- Bilirubin- Green on pale yellow background

    36

  • PORPHYRIN PIGMENTS

    Normally occur in tissues in small amounts.

    Considered to be precursor of the heme portion of Hb

    PORPHYRIAS are rare pathological conditions that are

    disorders of the biosynthesis of porphyrins and heme

    Found most abundantly in liver

    No method for demonstration other than Orange-red

    fluorescence which they give with UV light

    Porphyrins and bile pigments both give positive Gmelin

    reaction

    37

  • Porphyrin pigment-

    Appears as dense dark brown pigment

    In fresh frozen section exhibits a brilliant red fluorescence

    that fades rapidly with exposure to ultraviolet light.

    In paraffin sections and viewed using polarized light,

    shows as bright red in color with centrally located, dark

    maltese cross

    38

  • 39

  • MELANIN

    Melanin (melas= black), serves protective function- absorbs UV light

    In melanocytes, tyrosine DOPA Melanin

    Melanin is the brown-black, non-hemoglobin derived pigment

    Skin-

    It is synthesised in the melanocytes which are present in the basal cells of the

    epidermis

    Stored in the form of cytoplasmic granules in the phagocytic cells called the

    melanophages in inflammatory conditions, present in the upper dermis

    Benign nevus, Malignant melanoma

    40

    tyrosinase oxidase

  • Eye-

    Found in choroid, ciliary body, iris

    Melanomas (rare)

    Brain-

    In substantia nigra, macroscopically visible as black streak on both sides of

    mesencephalon

    Also in meninges (sooty appearance)

    Parkinsons disease this area is reduced

    41

  • DISORDERS OF PIGMENTATION

    HYPERPIGMENTATION:

    GENERALISED- Addisons disease, Chloasma

    FOCAL- Caf au lait spots, Peutz jeghers syndrome (peri oral),

    Melanosis coli, Melanotic tumors

    HYPOPIGMENTATION:

    GENERALISED- Albinism (tyrosinase activity of melanocytes genetically

    defective)

    FOCAL- Leucoderma ( form of partial albinism), Vitiligo

    ACQUIRED FOCAL- leprosy, healing of wounds, DLE, radiation

    dermatitis

    42

  • 43

    PERIORAL HYPERPIGMENTATION

  • 44

    ALBINISM VITILIGO

    HYPOPIGMENTATION

  • 45

  • 46

    MALIGNANT MELANOMA

  • METHODS FOR MELANIN

    Reducing methods- Fontana silver method, Schmrols

    reaction

    Enzyme methods

    Fluorescent methods

    Immunohistochemistry

    Solubility and bleaching characteristics

    47

  • MELANIN AND ITS

    PRECURSORS:

    Are capable of reducing both

    silver and acid ferricyanide

    solutions

    MELANINS:

    Completely insoluble in most

    organic solvents

    Bleached by strong oxidizing

    agents

    Powerful reducing agents

    48

  • REDUCING METHODS

    PRINCIPLE-

    Melanins argentaffin property- that is the reduction of

    ammonical silver solutions to form metallic silver without the

    need for a separate reducing agent.

    Melanin is also Argyrophilic, melanin is colored black by

    Silver impregnation methods

    Reduce ferricyanide to ferrocyanide with production of

    Prussian blue in the presence of ferric salts

    49

  • 50MASSON-FONTANA METHOD

  • 51

    MELANIN PIGMENT IN CELLS OF MALIGNANT MELANOMA,FONTANA-MASSON STAIN

  • SCHMORLS REACTION:

    Melanin Dark blue

    Nuclei Red

    LILLIES FERROUS ION UPTAKE REACTION:

    Melanin Dark green

    Nuclei Red

    LILLIES NILE BLUE METHOD:

    Melanin Dark blue

    Lipofuscin Dark blue

    Nuclei Red

    52

  • SECTION OF SKIN STAINED BY SCHMORLS REAGENT POSITIVE FOR

    MELANIN

    53

  • 54

  • 55

  • ENZYME METHODS

    Cells that are capable of producing melanin can be

    demonstrated by DOPA method

    These methods are those of

    Bloch and Laidlaw and Blackberg for tissue sections

    Bloch and Rodriguez and McGavran for tissue blocks

    58

  • SOLUBILITY AND BLEACHING METHODS

    Melanins are insoluble in organic solvents

    Due to tight bound it has with its protein component

    Use of strong oxidising agents will bleach melanin (slow, 16

    hours)

    Method of choice- Peracetic acid

    59

  • FORMALIN-INDUCED FLUORESCENCE

    Certain aromatic amines like 5-HT, Dopamine, Epinephrine,

    Norepinephrine, Histamine- show yellow fluorescence when

    exposed to formaldehyde

    Useful when demonstrating Amelanotic melanoma

    Results- Melanin precursor cell- Weak yellow fluorescence

    60

  • LIPID PIGMENTS/CHROMOLIPIDS

    Have lipid characteristics

    These are:

    1. Lipofuscins

    2. Ceroid

    3. Alcoholic hyaline

    4. Lipochromes

    5. Pseudomelanosis pigment

    64

  • LIPOFUSCINS

    Wear and tear pigment/ Brown atrophy pigments/ abnutzung pigments

    Produced by oxidation process of lipids and lipoproteins with aging

    Yellowish brown intracellular pigment

    M/E- coarse golden brown granular pigment, accumulates in central part

    of the cells around the nuclei.

    These are formed by slow progressive oxidation process, thus reactions

    vary according to the degree of oxidation present in the pigment

    65

  • Found in:

    Atrophied cells of old age

    Hepatocytes

    Cardiac muscle cell (brown atrophy of heart)

    Inner reticular layer of normal adrenal cortex

    Testis, in interstitial cells of Leydig (gives tissue brown color)

    Ovary

    Edge of cerebral haemorrhage or infarct

    66

  • 67

  • 68

  • 69

  • 70

  • 71

  • DEMONSTRATION OF LIPOFUSCINS

    Periodic acid- schiff method

    Schmorls ferric-ferricyanide reduction test

    Long ziehl-Neelsen method

    Sudan black B method

    Gomoris aldehyde fuchsin technique

    Masson-Fontana silver method

    Churukians silver method

    Lillies Nile blue sulfate method

    73

  • 74

  • CEROID

    It is a mixture of Lipofuscin like pigment, probably represents it in an

    early stage of formation

    Occurs in

    Atheroma

    Alcoholic cirrhosis

    Occurs as globules of yellow material within macrophages

    Differs from lipofuscin by negative Schmorl reaction

    Exhibits autofluorescence-

    greenish yellow in frozen sections

    Brownish yellow in paraffin sections

    75

  • ALCOHOLIC HYALIN

    Hyaline eosinophilic material, irregular to round mass

    (Mallory bodies) near the nuclei of liver cells in chronic

    alcoholics

    Represent enlarged, distorted, degenerated mitochondria

    Affinity towards acid fuchsin and eosin

    MALLORYS HEMALUM-PHLOXINE METHOD-

    Alcoholic hyalin- red

    Nuclei- blue

    76

  • 77

  • PSEUDOMELANOSIS PIGMENT

    Pseudomelanosis condition in which a dark brown, melanin-

    like pigment is found in macrophages in the mucosae of the

    large bowel and appendix

    Stains blue-green in the ferric-ferricyanide reduction test

    78

  • 79

    PSEUDOMELANOSIS PIGMENT

  • OTHER ENDOGENOUS PIGMENTS

    CHROMAFFIN:

    Normally found in adrenal medulla as dark brown, granular material. Occur in

    tumors of adrenal medulla- pheochromocytoma

    Demonstrated by Schmorls reaction, Lillies Nile blue A, the Masson-Fontana,

    PAS technique

    DUBIN-JOHNSON PIGMENT:

    Found in liver of patients of Dubin-Johnson syndrome- brownish black, granular,

    intracellular pigment, situated in the centrilobular hepatocytes

    HAMAZAKI-WEISENBERG BODIES:

    Small, yellow brown spindle shaped structures in sinuses of lymph nodes in

    patients with sarcoidosis

    80

  • EXOGENOUS PIGMENTS

    81

  • Introduced in the body by- Inhalation /

    Ingestion /Inoculation

    Broadly classified as

    1. Inhaled pigments (Carbon)

    2. Ingested pigments (Lead)

    3. Injected pigments (Tattooing)

    Majority of these pigments are infact colorless, some are

    inert and unreactive

    82

  • TATTOO PIGMENT

    Tattooing is a form of localized, exogenous pigmentation of the skin.

    Pigments like India Ink, Cinnabar, Carbon inocluated are phogocytosed

    by dermal macrophages, in which they reside for the remainder of the life.

    Pigments do not usually evoke any inflammatory response

    Examples-

    Tattooing by pricking the skin with dyes

    Prolonged use of ointments containing mercury

    Dirt left accidently in a wound

    83

  • 84

  • CARBON

    Most commonly seen mineral in tissue.

    Commonly found in lung and adjacent lymph nodes of urban dwellers and tobacco smokers

    MAIN SOURCE- Car exhausts, smoke from domestic and industrial chimneys.

    Black pigmentation of the lung (Anthracosis) is result of massive depostion of carbon in coal workers.

    Macroscopically lungs appears almost Black. Lung disease is known as Coal workers pneumoconiosis ( found in association with silica found with coal and other mineral ores)

    85

  • 86The pigment particles on inhalation are trapped by the thin film of mucus in the nose,

    pharynx, trachea and bronchi

    Small amount reaches alveoli and taken up by alveolar macrophages

    Some of the pigment-laden macrophages are coughed out via bronchi, while some settle in the

    interstitial tissue of the lung and in the respiratory bronchioles and pass into lymphatics to be

    deposited in the hilar lymph nodes

    Carbon is extremely unreactive and inert and fails to be demonstrated with the

    conventional histological stains and histochemical methods.

    The site and nature of carbon deposits make identification relatively easy.

    It may be confused with melanin deposition but treatment with bleaching agents will

    show carbon unaffected, whereas melanin will be dissolved.

  • 87

    ANTHRACOSIS LUNG

  • 88

  • SILICA

    In the form of silicates is associated with the majority of all

    mined ores, also abundant in stone and sand and industries

    involved in grinding stone or sand blasting.

    Mine workers inhale large quantities of silica that can give

    rise to the disease SILICOSIS.

    Silicosis consists of diffuse, nodular, whorled proliferation of

    fibrous tissue surrounding the tiny doubly refractile silica

    crystals when examined by polarized light.

    89

  • Silica is unreactive thus not demonstrated by histological

    stains and histochemical methods.

    It is anisotropic (birefringent) when examined using polarized

    light.

    HEMATITE LUNG-

    Mining hematite (ferric oxide) from quartz ores

    Silica from quartz play major role

    Iron in hematite lung fail to give Prussian blue reaction unless treated

    before with 40% HCl

    90

  • 91

  • 92

  • ASBESTOS

    Special form of silica used as a fire resistant and insulating material.

    Type of asbestos fibers that cause pulmonary disease are called AMPHIBOLES.

    Dangerous type is CROCIDOLITE. Fibers are 5-100 m long and only 0.25-0.5

    m in diameter and can collect in the alveoli at the periphery of lung.

    Fibers are anisotropic but fail to show birefringence when appear as asbestos

    body

    ASBESTOS BODY Characteristically beaded, yellow- brown, dumb-bell shaped

    in lung sections. The proteinaceous coat contains hemosiderin and is positive with

    Perls Prussian blue.

    93

  • 941) In case where asbestosis is suspected but no asbestos fibres or bodies are demonstrable

    lung tissue from lower lobes can be digested with 40% sodium hydroxide.

    Resultant tissue sludge is then centrifuged and washed in water

    Smears from the deposit are made and examined using polarized light

    2) Thick paraffin sections of lung tissues are mounted on glass slides coated with an adhesive

    The sections are dewaxed and mounted unstained and then examined using polarised light

    (Many thick sections may be needed before a positive result is seen)

    DEMONSTRATION OF ASBESTOS FIBRES

  • 95

  • 96

  • LEAD

    Environmental pollution due to Lead has been greatly reduced

    Lead pipes that carried much of the domestic water supply have been replaced and lead in

    paint, batteries and gasoline has also been reduced

    Lead poisoning cases are rare and usually diagnosed biochemically using the serum

    In chronic lead poisoning, excessive amounts can be deposited within many tissues,

    particularly bone and kidney tubules

    Demonstrated by various methods- Rhodizonate method (popular), Sulfide silver of

    Timm, the unripened hematoxylin technique of Mallory and Parker, but neither of these is

    specific for Lead.

    97

  • RHODIZONATE METHOD

    RESULTS:

    Lead salts Black

    Background Green

    98

  • 101

  • BERYLLIUM AND ALUMINUM

    BERYLLIUM is used in the manufacture of fluorescent light tubes and gains

    access to the body by inhalation or traumatization of the skin

    A foreign body granuloma is formed.

    These bodies usually give a positive reaction with Perls Prussian blue.

    ALUMINUM rarely seen in tissues but gains access to the body in similar

    way to Beryllium

    It can also be found in bone biopsies from patients on regular hemodialysis

    for chronic renal failure

    102

  • SOLOCHROME AZURINE METHOD FOR BERYLLIUM AND ALUMINUM

    RESULTS:

    Solution A: Aluminum and Beryllium Blue

    Solution B : Beryllium only Blue-Black

    Nuclei Red

    104

  • 105ALUMINON METHOD FOR ALUMINUM

    RESULTS-

    Aluminium Red

    Background Green

  • SILVER

    Rarely seen in the skin of silver workers as a result of

    industrial exposure

    Resultant permanent Blue-Gray pigmentation is called

    Argyria and is marked in Sun exposed areas

    Now commonly seen as localized change in mouth (amalgam

    tattoo)

    In unstained and H&E sections the silver appears as fine

    dark brown or black granules, particularly in basement

    membranes and sweat galnds.

    106

  • RHODANINE METHOD

    METHOD:

    Paraffin sections to distilled water

    Incubate sections in rhodanine solution at 37C for 24 hours

    Wash well in distilled water

    Mount in glycerin jelly

    RESULTS:

    Silver deposits Reddish- brown

    107

  • 108

    SIVER PIGMENT- RHODANINE METHOD

  • ARTIFACT

    PIGMENTS

    109

  • This group of pigments comprises:

    Formalin

    Malaria

    Schistosome

    Mercury

    Chromic oxide

    Starch

    110

  • FORMALIN PIGMENT

    a/k/a ACID HEMATIN, formed after several weeks in specimens by the interaction of acidic formaldehyde solutions with blood.

    Traces of formic acid are formed by oxidation, which decreases the quality of nuclear staining and leaches out hemosiderin resulting in formation of formalin pigment.

    COLOR- Brown or Brownish-Black deposit in tissues (product of degradation of hemoglobin, settles out as an insoluble product, extracelluarly)

    MORPHOLOGY- Vary, commonly seen as microcrystalline deposit that is anisotropic(birefringent).

    111

  • The deposit is usually present in blood rich tissues such as

    spleen, blood vessels, hemorrhagic lesions.

    Fixation of these organs for long period will tend to increase

    the amount of formalin pigment formed. Under these

    conditions it is advisable to change the fixative on regular

    basis.

    Use of Buffered neutral formalin will help to minimize the

    problem of formalin pigment deposition.

    112

  • 113

    Section of Kidney showing formalin pigment

  • REMOVAL OF FORMALIN

    PIGMENT BEFORE STAINING

    PICRIC ACID METHOD: Treat sections in saturated solutions of picric

    acid for 5 minutes to 2 hours.

    SCHRIDDES METHOD: Treat sections for 30 minutes with a mixture of

    200 ml of 75% alcohol and 1 ml of 25-28% liquor ammonia. Wash in

    water.

    VERCAYS METHOD: Treat sections for 10 minutes with a mixture of

    100 ml of 80% alcohol and 1 ml of aqueous potassium hydroxide. Wash

    in water.

    114

  • LILLIES METHOD: Treat sections for 1-5 minutes with a

    mixture of 50 ml of 75% acetone, 50 ml 3% hydrogen

    peroxide and 1 ml of 28% ammonia water followed by

    washing in 70% alcohol and then in running water.

    KARADASEWITSCHS METHOD: Treat sections for 30

    minutes to 1 hour with a mixture of 100 ml of 70% ethyl

    alcohol and 1 ml of 28% ammonia water. Wash in water.

    115

  • MALARIAL PIGMENT

    Morphologically similar to formalin pigment exhibits birefringence.

    It is formed within or in the region of RBC that contain the parasite

    The pigment sometimes may be so dense that can obscure the vision of

    parasite in RBC

    Pigment can also seen in the phagocytic cells that ingest the infected RBCs.

    Thus should examine for Kupffer cells of liver, the sinus lining of lymph nodes

    and spleen, and within phagocytic cells of bone marrow.

    116

  • 117MALARIAL PIGMENT

    IN PLACENTA IN SPLEEN

  • EXTRACTION OF MALARIAL PIGMENT

    REAGENT-

    Removed with saturated alcoholic picric acid for 12-24 hours

    PROCEDURE-

    1. Bring sections to water.

    2. Place the sections in alcoholic picric acid

    3. Rinse sections in 90% alcohol

    4. Rinse sections in 70% alcohol

    5. Place sections in tap water

    6. Stain with H&E or other routine stain

    118

  • MERCURY PIGMENT

    Pigment seen in the tissues that have been fixed in the mercury containing

    fixatives (B5, Heidenhains, Zenkers fluid)

    MORPHOLOGY-

    Varies, usually seen as Brownish black, extracellular crystal, monorefringent

    (birefringent when formalin fixed tissue has been secondarily fixed in formal

    mercury)

    120

  • 121

    SECTION OF KIDNEY SHOWING MERCURY

    PIGMENT

  • 122

    REAGENTS:

    1. Lugols iodine 1 g, Potassium iodide 2 g, distilled water 100 ml

    2. 5% aqueous Na2S2O3 (sodium thiosulphite)

    METHOD:

    1. Bring sections to water

    2. Place in Lugols iodine for 15 minutes

    3. Wash in water

    4. Place in thiosulfate for 3 minutes

    5. Wash in water

    6. Stain with H&E or other technique

    NOTE:

    Advisable to not remove mercury pigment with iodine solutions prior to staining with grams method as

    the connective tissue will take up crystal violet and then resist acetone decolorization.

  • 123SCHISTOSOME PIGMENT:

    Occasionally seen in tissue sections infested with Schistosomes.

    Pigment tends to be chunky, properties similar to formalin and malarial pigments.

    CHROMIC OXIDE:

    Pigment rarely seen. Presents as fine yellow-brown particulate deposit,

    monorefringent and extracellular in tissues fixed in chromic acid or dichromate

    containing fixatives.

    Removed from tissues by treatment with 1% acid alcohol.

    STARCH:

    Pigment introduced by talcum powder from the gloves of surgeon/ nurses/

    pathologists.

    It is PAS and Gomori Methamine Silver Positive and produce Maltese cross

    configuration when polarised.

  • REFERENCES

    1. Kumar v, Abbas K A, Aster C J. Cellular responses to stress and toxic insults: Adaptation, Injury and death. In: Kumar v, Abbas K A, Aster C J, editors. Robbins and cotran pathological basis of disease. 9th ed. New delhi : Elsevier ; 2014

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