Pigments &

Minerals
1. Endogenous Pigments

 Substances produced either within tissue and serve a

physiological functions
 Or are by-products of normal metabolic processes
 Subdivisions
 Hematogenous pigments
 Non-hematogenous pigments
 Endogenous minerals

E.M.M.MANGADA, RMT 2
2. Artifact Pigments

 Deposits of artifactually produced material cause by the

interactions between certain tissue components and some
chemical substances
 Example: malaria and formalin

E.M.M.MANGADA, RMT 3
3. Exogenous Pigments and Minerals

 Gain access to the body accidentally and serve no

physiologic function
 Entry is gained by inhalation into the lungs or by
impantation into the skin
 Example: minerals which are pigmented

E.M.M.MANGADA, RMT 4
Consideration before carrying out Various
stains and histochemical reactions

 Pigment’s morphology
 Tissue site
 Relevant clinical data

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 ENDOGENOUS
PIGMENTS
HEMATOGENOUS

NON-HEMATOGENOUS PIGMENTS
ENDOGENOUS MINERALS

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Hematogenous Pigments
Hemosiderins

Hemoglobin

Bile pigments
Porphyrins

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Hemosiderins

 Seen as yellow or brown granules and normally

appear intracellularly
 Contain iron in the form of ferric hydroxide that is
bound to a protein framework and is unmasked by
various chemicals

E.M.M.MANGADA, RMT 8
DEMONSTRATION OF
HEMOSIDERIN
PERL’S PRUSSIAN BLUE
LILLIES METHOD FOR FERRIC AND FERROUS IRON
HUKILL AND PUTT’S METHOD

E.M.M.MANGADA, RMT 9
Demonstration of Hemosiderin
and Iron
 After fixation in formalin, hemosiderin becomes soluble
with dilute acids, especially oxalic acids
 Fixatives that contain acids can give a negative reaction
for iron pigment
 Can be demonstrated in Perl’s Prussian blue if treated
with hydrogen peroxide or if the ferrocyanide is heated to
60C in water bath, oven, or microwave oven
 However, use of heat will sometimes cause a fine blue
precipitate
E.M.M.MANGADA, RMT 10
Perl’s Prussian Blue

 Considered to be the best first classical histochemical

reaction
 Treatment with acid ferrocyanide solution will resultin
unmasking of ferric iron in the form hydroxide by dilte HCl
 The ferric iron reacts with a dilute potassium ferrocyanide
solution to produce an insoluble compound, ferric
ferrocyanide (Prussian blue)

E.M.M.MANGADA, RMT 11
Perl’s Prussian Blue

 Fixation: avoid use of acid fixatives

 Chromates will also interfere with the preservation of iron
 Sections: works well on all type of section, including resin
 Results: ferric iro-blue; nuclei-red

E.M.M.MANGADA, RMT 12
Lillie’s Method

 Fixation: avoid the use of acid fixative

 Sections: paraffin, frozen and resin
 Results: ferric iron- dark Prussian blue; ferrous
iron- dark Turnbull’s blue; nuclei- red

E.M.M.MANGADA, RMT 13
Hukill and Putt’s Method

 Fixation: not critical but avoid prolonged

exposure to acid fixative
 Sections: all types of sections including resin
 Results: ferrous iron- red; nuclei- blue

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DEMONSTRATION OF
HEMOGLOBIN
LEUCO PATENT BLUE V METHOD

E.M.M.MANGADA, RMT 15
Demonstration of Hemoglobin

 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

E.M.M.MANGADA, RMT 16
Leuco Patent Blue V Method
 Intorduce by Lison (1938) and later was modified by Dunn and Thompson
(1946]
 Fixation: Formalin or formal mercury
 Results: hemoglobin peroxidase (RBCs and neutrophils)- dark blue; Nuclei-red

E.M.M.MANGADA, RMT 17
DEMONSTRATION OF BILE
PIGMENTSAND HEMATOIDIN
 MODIFIED FOUCHET’S TECHNIQU FOR LIVER BILE PIGMENTS
GMELIN TECHNIQUE

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Demonstration of Bile Pigments
and Hematoidin
 The need to identify bile pigments arises mainly in the
histological examination of the liver, where lipofuscin may
be of significant importance
 Both appear yellow-brown in H&E –stained paraffin
section
 Green color of biliverdin is often masked with esoin
 Bile pigments are not autofluorescent;lipofuscin is
autofluorescent

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Modified Fouchet’s technique for
Liver Bile Pigments
 Commony used method for demonstration of bile pigments
 Pigments is converted to green color of biliverdin and blue
cholecyanin by the oxidative action of ferric chloride in
the presence of TCA
 Quick and simple to carry out and when counterstained
with Van Gieson’s solution of green color is accentuated

E.M.M.MANGADA, RMT 20
Modified Fouchet’s technique for
Liver Bile Pigments
 Fixation: any fixative appears suitable
 Sections : Any
 Results: Bile pigments- emerald to blue-green; muscle-
yellow; Collagen-red

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Gmelin Technique

 The only method that shows an identical result with liver

bile, gallbladder bile, and hematoidin
tends to be messy, capricious, and gives impermanent
results
 Deparaffinized tissue containing bile pigments are treated
with nitric acidand a changing color spectrum is produced
 It is unreliable- it is advisable to repeat the test for at
least three times
 Popular modification is the used of bromine in carbon
tetrachloride as an oxidant
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Gmelin Technique

 Sections: Paraffin
 Results: Bile pigments will gradually produce the following
spectrum of color change: yellow-green-blue-purple-red
 The method is impermanent
 The reaction can occur rapidly but can be slowed down
using 50-70% solution of nitric acid

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Demonstration of Porphyrin
Pigments
 These substances normally may occur in the tissues in only
small amounts
 Considered to be precursors of the heme portion of
hemoglobin
 Porphyrias are rare pathological conditions that are
disorders of the biosynthesis of porphyrins and heme

E.M.M.MANGADA, RMT 24
Demonstration of Porphyrin
Pigments
 In erythropoietic porphyria, porphyrin appears as a dense
dark brown pigment and in fresh frozen sections exhibits a
brilliant red fluorescence that rapidly fades with exposure
to UV
 In paraffin section viewed in polarized light, shows a
bright red color with centrally located dark Maltese cross

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Non-hematogenous Pigments
Melanins Dubin-Johnson pigment
Lipofuscins Ceroid-type lipofuscins
Chromaffin Hamazaki-Weisenberg bodies
Pseudomelanosis (melanosis coli
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Melanins

 Groups of pigments which color varies from light brown to black

 Normally found in the skin, eye, substantia nigra of the brain and hair
follicles
 These are bound to proteins and localized in the cytoplasm of cells
within “melanin granules”
 Common sites where melanin can be found:
 Skin
 Eye
 Brain

E.M.M.MANGADA, RMT 27
Demonstration of Melanin and
Melanin-producing Cells
Reducing methods such as the Masson-Fontana silver Technique and Schmorl’s ferric
Ferricyanide reduction test
Enzyme methods
Solubility and bleaching characteristics
Flurorescent methods
Imunohistochemistry

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Reducing methods for
Melanin

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Masson-Fontana method for
melanin
 Fixation: formalin is best; chromate and mercuric
chloride should be avoided
 Sections: Works on all types of section, although some
adjustment is necessary for resin section
 Results: Melanin, argentaffin, and chromaffin and some
lipofuscins- black; Nuclei- red

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Microwave Ammoniacal Silver method
for argentaffin and melanin
 Fixation: 10% NBF
 Results: Melanin, Argentaffin, chromafifin, lipofuscins
and other silver reducing substances- black; nuclei-red
 Note: Results obtained with this method are similar to
those obtained with the Masson-Fontana technique

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Schmorl’s Reaction

 Fixation: 10% NBF

 Results: Melanin, argentaffin cells, chromaffin
some lipofuscins, thyroid colloid- dark blue;
Nuclei- red

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Enzyme methods

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DOPA Method
 Demonstrates cells capable of producing melanin
 The enzyme tyrosinase localized within the cells
will oxidize DOPA to form an insoluble brown-
black pigment
 Best results are obtained using post-fixed cryostat
sections

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DOPA Oxidase Method
 Method not in use
 Enzyme tyrosinase in cells will oxidize DOPA to
form an insoluble brown-black pigment
 By Bloch (1917) and Laidlaw and Blackberg (1932)
for tissue sections
 By Bloch (1917) and Rodriguez and McGavran
(1969) for tissue blocks

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Solubility an Bleaching
Methods

E.M.M.MANGADA, RMT 36
Solubility and Bleaching
Methods
 Uses strong oxidizing agents such as permanganate,
chlorate, chromic acid, peroxide, and peracetic acid that
will bleach melanin
 Process is slow taking 16 hours
 Lipofuscins take longer time to be bleached from paraffin
sections than melanin
 Method of choice: peracetic acid but treatment with
0.25% potassium permanganate followed by 2% oxalic acid
also works well
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Formalin-induced
fluorescence

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Formalin- induced
Fluorescence
 Particularly useful in demonstrating amelonitc melanoma
 Melanin precursors present will form a product of
isocarboline derivatives that are dehydrogenated and will
show yellow fluorescence
 Best result are seen when using freeze-dried sections and
then fixed using parafromaldehyde vapor

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Formalin- induced
Fluorescence
 Fixation: 10% NBF
 Sections: Cryostat, or 5-um paraffin sections
 Results: Melanin precursor cells- weak yellow
fluorescence

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Other Methods for
Formalin

E.M.M.MANGADA, RMT 41
Ferrous Iron Uptake Reaction for
Melanin
 Fixation: Formalin is best; avid all chromate fixatives
 Sections: Paraffin
 Results: Melanin of skin, eye, pin and neuromelanin- dark
green; Nuclei- red
 Notes: according to Lilies this method is specific for
melanin
 Ferric iron and lipofuscin do not stain with this method

E.M.M.MANGADA, RMT 42
Nile blue method for melanin and
lipofuscin
 Fixation: 10% NBF
 Sections: paraffin
 Results: melanin- dark blue; lipofuscin- dark
blue; Nucei- blue or unstained
 Notes:
 Using frozen sections, this method will stain eutral
lipids red to pink
 Acidic lipids stain blue
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Immunohistochemistry
 Uses antibodies to demonstrate melanocytic
lesions
 More widely use antibodies: S100, HMB-45,
melanin A and to lesser extent PGP9.5
 HMB-45- demonstrate melanosome formation and
melanocytic differetiation

E.M.M.MANGADA, RMT 44
Lipofuscins

 Yellow-brown to reddish-brown pigments occur widely

throughout the body and are thought to be produced by
an oxidation process of lipids and LPP
 Found in the following sites: hepatocytes, cardiac muscle
cells, adrenal cortex, testis, ovary, CNS, bone marrow,
cervix, kidney etc.

E.M.M.MANGADA, RMT 45
Demonstration of
Lipofuscins
Periodic-acid Schiff’s method Gomori’s aldehyde fuschin technique
Schmorl’s ferric-ferricyanide reduction test Masson-Fontana silver method
Long-Ziehl-Neelsen method Basophilia using methyl green
Sudan Black B method Churukian’s silver method

E.M.M.MANGADA, RMT Lilie’s Nile blue sulfate method 46

Long Ziehl-Neelsen method

 Fixation: any
 Sections: works well on all types of tissue section
 Results: lipofuscins- magenta; ceroid- magenta; nuclei-
blue; background- pale magenta to pale blue

E.M.M.MANGADA, RMT 47
Aldehyde Fuschin Technique

 Fixation: 10% NBF

 Sections: Paraffin
 Solutions: acidified potassium permanganate
solution
 Results: Lipofuscins- purple; elastic- purple

E.M.M.MANGADA, RMT 48
Chromaffin

 Normally found in the cells of adrenal medulla as dark

brown, granular material
 May occur in pheochromcytoma
 Fixation: Orth’s or dichromate containing fixatives are
recommended
 May be demonstrated by Schmorl’s reaction, Lilie’s Nile
blue A, Masson-Fontana, Churukian’s microwave
ammoniacal silver method and PAS technique

E.M.M.MANGADA, RMT 49
Pseudomelanosis pigment
(melanosis cell)
 Sometimes seen in macrophages in the lamina
propria of the large intestine and appendix
 Stain in methodsvthat are used to dmeonstrate
lipofuscin such as Masson-Fontana and Schmorl’s

E.M.M.MANGADA, RMT 50
Dubin-Johnson pigment

 Found in the liver of patients with Dubin-Johnson

syndrome and is due to defective canalicular transport of
bilirubin
 Characterized by presence of brown-black, granular,
intracellular pigment situated in the centrilobular
hepatocytes
 Histochemically similar to lipofuscin

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Ceroid-type lipofuscins

 Fail to stain with ferric-ferricyanide

 A lipofuscin in early stage of oxidation

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Hamazaki-Weisenberg Bodies

 Small, yellow-brown spindle-shaped structures found

mainly in the sinuses of lymph nodes
 Seen in patients with sarcoidosis and associated with
melanosis coli
 Histochemically similar to lipofuscin
 Ultrastructural level suggest probably are giant lysosomal
residual bodies

E.M.M.MANGADA, RMT 53
Endogenous Minerals
Calcium

Copper

Uric acid and urates

E.M.M.MANGADA, RMT 54
Calcium

 Abnormal depositions can be found in necrotic areas of

tissue associated with tuberculosis, infarction (Gandy-
Gamma bodies), atheroma in blood vessels , and
malakoplakia of the bladder (Michaelis-Gutman bodies)
 Calcium salts are usually monorefringent and calcium
oxalate is birefringent
 Calcium usually stains purple blue with H&E
 Dyes used for demonstration of calcium: alizarin red S,
purpurin, naphthochrome green B, and nuclear fast red
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Calcium

 Von Kossa with silver nitrate- preferred for

demonstration of calcium in paraffin sections

E.M.M.MANGADA, RMT 56
Alizarin red S method for calcium

 Fixation: NBF, formal alcohol and alcohol

 Sections: Paraffin and frozen
 Results: Calcium deposits- orange-red; background- green

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Copper

 Mallory’s unrepined hematoxylin- copper forms

blue dye lalke
 Other methods include the rubeanic method for
copper, modfied rhodamine technique

E.M.M.MANGADA, RMT 58
Copper

 Accumulation is associated with Wilson’s disease

 Kayser-Fleischer ring – brown ring of deposited
copper in the cornea
 Deposition is also associated with primary biliary
cirrhosis and certain hepatic disorderer

E.M.M.MANGADA, RMT 59
Rubeanic acid method for Copper

 Fixative: 10% NBF

 Results: copper- greenish black; nuclei- pale red

E.M.M.MANGADA, RMT 60
Modified Rhodamine Technique

 Fixative: 10% NBF

 Section: Paraffin
 Results: copper and copper associated protein-
red to orange-red; nuclei- blue; bile-green

E.M.M.MANGADA, RMT 61
Uric Acid and urates

 Breakdown products of body purine metabolism; small

proportion is obtained in the diet
 High levels result in deposition, which are water soluble in
tissues causing:
 subcutaneous nodular deposits of urate crystals (‘tophi’)
 Synovitis and arthritis
 Renal disease and calculi

E.M.M.MANGADA, RMT 62
 ARTIFACT
PIGMENTS
FORMALIN

MALARIA

SCHISTOSOME

MERCURY

CHROMIC OXIDE
STARCH
E.M.M.MANGADA, RMT 63
Formalin Pigment
 Seen as brown or brownish-black depsosit in tissues that
have been fixed in acidic formalin
 Usually seen in blood-rich tissues such as spleen,
hemmorhagic lesions, and large blood vessels filled with
blood
 Morphology of pigment is commonly seen as
microcrystalline deposit that is anisotropic (birefringent)
 One way of removing pigment is by treating unstained
tissue sections with sat. alcoholic picric acid
E.M.M.MANGADA, RMT 64
Formalin Pigment
 Seen as brown or brownish-black depsosit in tissues that
have been fixed in acidic formalin
 Usually seen in blood-rich tissues such as spleen,
hemmorhagic lesions, and large blood vessels filled with
blood
 Morphology of pigment is commonly seen as
microcrystalline deposit that is anisotropic (birefringent)
 One way of removing pigment is by treating unstained
tissue sections with sat. alcoholic picric acid or 10%
ammonium hydroxide in 70% alcohol for 5-15 minutes
E.M.M.MANGADA, RMT 65
Malarial Pigment

 Morphologically similar to formalin pigment

 Formed within or in the region of RBCs with the malarial
parasite
 Can be seen over the RBC within tiny blood capillaries of
the brain in infection with P. falciparum
 Like formalin pigment, exhibits birefringence and can be
removed with sat. alcoholic picric acid within 12-24 hr

E.M.M.MANGADA, RMT 66
Schistosome Pigment
 Occasionally seen in tissue sections where infections with
Schistosoma can be seen
 Chunky and shows similar properties to both formalin and
malarial pigments

E.M.M.MANGADA, RMT 67
Mercury Pigment

 Seen in mercury-containing fixatives

 Usually monorefringent
 In long storage of tissues, pigment changes from
crystalline form to globular one (exhibits a Maltese cross
birefringence)
 Do NOT remove pigment with iodine solutions which
cause CT to take up crystal violet and resist acetone
declorization

E.M.M.MANGADA, RMT 68
Chromic Acid

 Rarely seen in tissues

 Present as fine yellow-brown particulate deposit
in tissue
 May be reduced using graded alcohol
 Monorefringent and extracellular
 Can be removed by 1% acid alcohol

E.M.M.MANGADA, RMT 69
 EXOGENOUS
PIGMENTS & MINERAL
LEAD

SILVER

E.M.M.MANGADA, RMT 70
Lead
 Can be deposited within many tissues,
particularly bone and kidney tubules
 Rhodizonate method- most popular method for
demonstration of lead deposits
 Nonspecific methods: sulfide-silver and
unripened hematoxylin technique

E.M.M.MANGADA, RMT 71
Rhodizonate method for Lead
salts
 Fixation: avoid use of mercuric-containing
fixatives
 Sections: Paraffin
 Results: lead salts- black; background- green

E.M.M.MANGADA, RMT 72
Silver

 Rarely found in the skin

 More commonly seen as localized change in the mouth
 Appear fine dark brown or black granules in unstained and
H&E sections, particulary in basement membrane and
sweat glands

E.M.M.MANGADA, RMT 73
Rhodanine method for silver

 Fixation: not critical but avoid using mercury-containing

fixatives
 Sections: paraffin; frozen sections (best results)
 Results: silver deposits- reddish-brown

E.M.M.MANGADA, RMT 74