Staining Pigments and Deposits

TISSUE PIGMENTS AND DEPOSITS
PIGMENTATION
• Endogenous
• Hematogenous
• Non-hematogenous
• Endogenous minerals
• Exogenous
• Artefacts
ENDOGENOUS PIGMENTS
HEMATOGENOUS PIGMENTS
• Naturally formed within the tissue due to breakdown of Hgb

caused by excessive blood destruction
• HEMOSIDERIN
• yellow to brown granule
• Free iron in the form of ferric hydroxide bound to protein
complexes
• Found in places accessible to oxygen
• Most common Hgb derivative
• Removed by acids
• HEMATOIDIN
• Iron free pigment
• Found in poorly oxygenated places
• Formation of bile pigments
• Bright yellow in old splenic infarcts
• Old hemorrhagic areas in the brain
• HEMATIN
• Minus globin molecule
• Old blood clots
• Malaria, pernicious anemia and toxic hemolysis
• HEMOZOIN
• Black granule seen in malaria
• Removed by alcoholic picric acid
• HEMOSFUSCIN (LIPOFUSCIN)
• Iron free brownish yellow pigment
• With hemosiderin in hemochromatosis
• Does not react with ferrocyanide but stains intensely
with basic dyes
PERL’S PRUSSIAN BLUE
• For hemosiderin (ferric iron)

• Unmasking ferric iron by dilute HCl (a component of the acid
ferrocyanide solution
• Ferric iron + dilute potassium ferrocyanide = ferric ferrocyanide
• RESULTS
• Hemosiderin and ferric salts deep blue
• Other pigments retain natural color
• Tissues and nuclei red
GOMORI’S PRUSSIAN BLUE
• For IRON
• RESULTS
• Iron pigments bright blue
• Nuclei red
• Cytoplasm pink to rose
TURNBULL’S BLUE REACTION
• For ferrous iron (hemosiderin)

• Ferrous iron + potassium ferricyanide = ferrous
ferricyanide
• RESULTS
• Hemosiderin blue
• Nuclei red
BENZIDINE NITROPRUSSIDE
• For hemoglobin and oxidase granules

• Ferrous iron + potassium ferricyanide = ferrous ferricyanide
• RESULTS
• Hemoglobin and some oxidase granules dark blue
• Nuclei red
• Other tissue components faint pink
BILE PIGMENTS AND HEMATOIDIN
• Both conjugated and unconjugated bilirubin, biliverdin and

hematoidin
• H&E : small yellow brown globules
• Must be distinguished from lipofuscin (autofluorescent) but
both are positive with Scmorl’s ferric ferricyanide
reduction test.
FOUCHET TECHNIQUE
• For bile pigment

• Bile pigment is converted to the color of biliverdin (green)
and blue cholecyanin by oxidative reaction of the ferric
chloride in the presence of trichloroacetic acid
• RESULTS :
• Bile pigments emerald to blue green
• Muscle yellow
• Collagen red
GMELIN TEHCNIQUE
• Shows an identical result with liver bile, gallbladder bile,

and hematoidin
• Diagnostic for bile pigments
• Bilirubin is oxidized to blue green biliverdin by nitric acid
• Yellowish brown  blue and purple  green
• If negative, repeat test 3x
• Oxidizing agents ferric salts, iodine and potassium
bichromate
STEIN’S IODINE TEST
• For bile pigment

• Depend upon the oxidation of the pigment to
green biliverdin by iodine
SCHMORL’S FERRIC FERRICYANIDE
METHOD
• Reduction of ferricyanide to ferrocyanide with the
production of prusssian blue in the presence of ferric
salts (Schmorl’s reaction)
• RESULTS
• Bile, lipofuscins, melanin dark blue
• Argentaffin, chromaffin dark blue
• Thyroid colloid dark blue
• Nuclei red
GOMORI’S ALDEHYDE FUCHSIN
• For lipofuscin
• Paraldehyde should be freshly opened.
• RESULTS :
• Lipfuscin purple
• Background yellow
MALLORY’S FUCHSIN STAIN
• For hemofuscin
• RESULTS :
• Nuclei blue
• Hemofuscin red
• Hemosiderin unstained
MELANIN
• An autogeous pigment (brown / black)

• Skin and eyes
• Tyrosine  DOPA  melanin
• Soluble in strong alkali , does not react with Fe and fat
stain
• Nevus
MASSON FONTANA SILVER TECHNIQUE
• For MELANIN and argentaffin cells

• Melanin reduces ammoniacal silver without the use of
extraneous reducer (argentaffin reaction)
• RESULTS :
• Melanin & argentaffin cells black
• Nuclei red
CALCIM DEPOSITS
• Normally found in bones and teeth

• Pathologic : calcium salts of phosphates and carbonates in
tissue
• H&E: deep purplish blue
• SOLUBLE CALCIUM SALTS
• Non ionized / non bound
• Gypsum method  Ca + sulfuric acid = gypsum crystals
• Oxalate method
CALCIM DEPOSITS
• INSOLUBLE CALCIUM SALTS

• Soluble in weak acids
• Fix in neutral solution
• CALCIUM DYE LAKE REACTION
• Calcium salts reddish orange
• Back ground pale green
• METAL SUBSTITUTION
• VON KOSSA SILVER NITRATE
• CALCIUM SALT IS BLACK, nuclei is red
COPPER
• Wilson’s disease
• Liver of patients with biliary cirrhosis
• Blue dye lake with alum hematoxylin
LINDQUIST MODIFIED RHODANINE
TECHNIQUE
• Method of choice for demonstrating copper and copper
associated protein
• Dimethyl aminobenzylidine rhodanin
• Apathy’s medium is the preferred mountant
• RESULTS :
• Copper red to orange red
• Nuclei blue
• Bile green
URATES AND PYROPHOSPHATES
• Monosodium urates – gout , - bifrengence

• Calcium pyrophosphate – chondrocalcinosis , +
bifrengence
• Urates can extracted by saturated aqueous lithium
carbonate, pyrophosphate are not
EXOGENOUS PIGMENTS
CARBON
• Most commonly seen exogenous mineral in tissues

• Anthracosis
• Coal worker’s pneumoconiosis
• Cannot be demonstrated, may be confuse with melanin
• Melanin is dissolved by bleaching agents, CARBON IS
NOT.
ARTEFACT PIGMENTS
• Due to faulty staining

• ACID ALCOHOL – most common solution for removing
• May be deposited during fixation
ARTEFACT PIGMENTS
FORMALIN
• Fine, dark brown or black crystal like
• In post mortem and blood containing tissues fixed with
acid formaldehyde
• To prevent : neutral or buffered formalin
• To remove : treat unstained sections with saturated
alcoholic picric acid or :
• Use 10% sodium hydroxide in absolute alcohol (10 mins )
• Treat with 10% ammonium hydroxide in 70% OH (5-15 mins)
ARTEFACT PIGMENTS
MERCURIC CHLORIDE
• Black / brown / grayish black granules
• Rarely in HEIDENHAIN’S SUSA
• To remove :
During dehydration  add few drops of saturated
alcoholic iodine
After sectioning  bleach with a weak sodium
thiosulfate
ARTEFACT PIGMENTS
OSMIUM TETROXIDE
• Improperly washed out black deposits
• To remove : BLEACH 
• Hydrogen peroxide and 70% OH
• 0.5% potassium permanganate
CHROME DEPOSITS
• Fine brown / black granules in chromate fixed tissues
• To remove : wash with ACID ALCOHOL

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Staining Pigments and Deposits

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TISSUE PIGMENTS AND DEPOSITS

• Naturally formed within the tissue due to breakdown of Hgb

• For hemosiderin (ferric iron)

• For ferrous iron (hemosiderin)

• For hemoglobin and oxidase granules

• Both conjugated and unconjugated bilirubin, biliverdin and

• For bile pigment

• Shows an identical result with liver bile, gallbladder bile,

• For bile pigment

• An autogeous pigment (brown / black)

• For MELANIN and argentaffin cells

• Normally found in bones and teeth

• INSOLUBLE CALCIUM SALTS

• Monosodium urates – gout , - bifrengence

• Most commonly seen exogenous mineral in tissues

• Due to faulty staining

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