PIGMENTS AND PIGMENT

DISORDERS

BY- DR SUCHITA MISHRA

(TUTOR, PATHOLOGY
DEPARTMENT, NHLMMC)
PIGMENTS-pigments are coloured
substance some are normal
constituents,other are abnormal
&get deposited in pathologic
conditions only.
 CLASSIFICATION
1.Endogenous Pigments
Haematogenous-Hemosiderin
Bile pigments
Hemoglobin
Porphyrins
Non-Haematogenous-
Melanin
Derived from Fat –
Lipofuscins , Ceroid pigment
Myoglobin
Dubin-Johnson pigment
Endogenous minerals/deposit-
Iron,Calcium,Copper,Urates
2.Artefact Pigments: Formalin,Malaria,Schistosomes,
Mercury,Chromic oxide,starch.
3.Exogenous Pigments & Minerals
By ingestion-Metals & Non metals
By inhalation-Coal,Silica,Asbestos.
By Injection-Tattoo.
HEMOSIDERIN

Normally it is seen as yellow to brown

intracellular pigments.
Contain iron in the form of ferric
hydroxide that is bound to a protein
framework.
Normally small amount can be seen in
mononuclear phagocytic cells of BM,liver
&spleen.
 IRON METABOLISM

Total body iron– 6gm(M) & 2gm(F).

Distribution:
80% of functional iron is found in Hb &
15-20% of total body iron is stored as ferritin
or hemosiderin.
No regulatory pathway-
lost by shedding of mucosal & skin
epithelium.
Balance---by regulating absorption.
 Dietery iron 1-2 mg/day
Iron excretion in nails,
Hairs , urine & feces
Duodenal and jejunal
absorption of iron
Fe transported to
Body tissues
Fe in Plasma

Plasma
transferrin
Bone marrow Ineffec
tiv e eryth
ropoies
hemopoiesis is Storage pool – bone marrow
Heme synthesis

Red cells Death of red cells

1700-2400 mg Fe released
(100-120 days)
 REGULATION
Iron absorption depends on body iron stores & regulated by
hepcidin.

HEPCIDIN-A small liver derived plasma peptide encoded by

the HMP gene.
Lower plasma iron level by binding with iron efflux channel
ferriportin.

Proteins regulating hepcidin level-

Hemojuvelin
Transferrin receptor 2
HFE
 HEMOSIDEROSIS
Two types-1.Localised haemosiderosis

2.Generalised haemosiderosis
Parenchymatous deposition
a.Haemocromatosis
b.Chronic haemolytic anaemia
c.Due to increased iron content in diet

Deposition in the mps-due to parental

iron administration or repeated blood trasfusions.
LOCALISED HEMOSIDEROSIS

Occur whenever there is a haemorrhage,the

iron from the broken red cells is found in
the nearby macrophages in the form of
haemosiderin.This is seen in-
a.organising haematoma
b.haemorrhagic infarcts
c.pulmonary
haemosiderosis(MS,LVF,Good pasture’s
syndrome)
 HEMOCHROMATOSIS
Autosomal recessive disorder,characterised by excessive
iron absorption from gut and subcequent accumulation
in different organs like liver,pancreas,heart.
Cause-mutation of HFE gene(type 1)
 Juvenile hemocromatosis-mut.hepcidin(type2a)
 mut. Hemojuvelin(type2b)
 mut.of transferrin receptor2(type3)
• mut.of ferroportin 1 gene(type4)

• Excess Iron is toxic to host tissue by-

a) Lipid peroxidation via iron catalysed free radical
reaction.
b) stimulation of collagen formation.
c) Interaction of reactive O2 species & of iron itself
with DNA.
 Morphological Changes
• LIVER
Hemosmiderin granules are deposited first in cytoplasm of periportal hepatocytes.

• PANCREAS
Intensively pigmented.
Diffuse interstitial fibrosis.
Parenchymal atrophy.
Hemosiderin is found in Acinar cells, Islet cells & in Interstitial fibrous stroma.

• HEART
Often enlarged.
Hemosiderin granules seen within myocardial fibres, producing brown
discolouration.

• SKIN
Hemosiderin is deposited in dermal macrophages & fibroblasts.
Pigmentation mainly due to increased epidermal melanin production.
Combination of Melanin & Hemosiderin gives Slate grey colour.
 DEMONSTRATION OF SIDEROSIS

• Best demonstrated by the PERL’S Stain (using acid

ferrocyanide), which gives PRUSSIAN BLUE reaction with
Hemosiderin.

• Simple grading of stored tissue iron includes grades from

1(minimal) to 4(massive deposits) with grades 2 & 3
indicating intermediate amounts.

• Hepatic iron index(HII) , represents the chemically

measured hepatic iron concentration ( mic.mol/g dry wt.)
divided by the pt’s age. It distinguishes genetic
hemochromatosis from other causes of siderosis.
HEMOSIDERIN GRANULES IN
HEPATOCYTES

H&E STAIN PRUSSIAN BLUE STAIN

 BILE PIGMENTS

• Bile pigments include both conjugated & unconjugated

bilirubin, biliverdin & hematoidin.

• Bilirubin is the end product of heme degradation.

• 85% of bilirubin is derived from breakdown of

senescent RBCs.

• Small amount derived from hemoproteins like P- 450

cytochromes.
BILIRUBIN METABOLISM
 JAUNDICE & CHOLESTASIS
• Alterations in bilirubin production & clearance may
lead to

a) Jaundice - yellow discolouration of skin & sclera due

to retention of bilirubin.

b) Cholestasis – characterised by systemic retention of

bilirubin & accumulation of bile pigment in hepatic
parenchyma.

Unconjugated bilirubin may diffuse in tissues like

the brain in infants & cause toxic injury
(Kernicterus).
 MORPHOLOGY IN CHOLESTASIS
Depends on severity and duration of underlying
cause.
There is accumulation of bile pigment in hepatic
parenchyma
Droplets of bile pigment is with in hepatocytes,which
take a fine foamy appearance(feathery degenaration)
There is bile ductular proliferation and periportal
oedema.
Neutrophilic infiltration surrounding hepatocytes
Ultimately the hepatocytes undergoing degenaration
and lead to billiary cirrhosis.
INTRACELLULAR CHOLESTASIS
DEMONSTRATION OF BILE PIGMENTS

• In early stages of Cholestasis, bile in hepatocytes appear

as green brown globules & then within bile canaliculi as
large smooth round rod or globules in H & E stain.

• Demonstration – by modified Fouchet technique in

which the pigment is converted to green colour of
BILIVERDIN & blue CHOLECYANIN by oxidative
action of ferric chloride.

• Gmelin Technique - Addition of conc. HNO3 on tissue

section causes colour change: yellow – green – blue –
purple - red.
 HEMOGLOBIN
• A conjugated protein, composed of a colourless protein
globin & a red pigmented component-heme.

• Heme is composed of protoporphyrin combined with

ferrous iron.

• Hemoglobin must be demonstrated in pathological cond.

like casts in renal tubules in cases of hemoglobinuria or
active GN.

• DEMONSTRATION: Hemoglobin is detected in tissue

sections by demonstrating the enz. Hemoglobin peroxidase
by the PATENT BLUE method where it appears dark blue.
 PORPHYRINS
• Porphyrins are considered to be precursors
of the heme portion of hemoglobin.

• Normally these substances occur in tissues

in small amounts.
 PORPHYRIN PIGMENT
• PORPHYRIAS are rare inherited disorders of specific enzymes in the
heme biosynthesis pathway.

• Types- Hepatic or Erythropoietic depending on the primary site of

overproduction & accumulation of the porphyrin precursors &
porphyrin.

• DETECTION- Porphyrin pigment can be seen as focal deposits in liver

section.

• It appears as a dense dark brown pigment.

• In fresh frozen section- exhibits a brilliant red fluorescence that rapidly

fades on exposure to ultraviolet light.

• In paraffin sections & viewed using polarized light, appears as bright red
in colour with a centrally located dark Maltese cross.
PROTOPORPHYRIN IN HEPATOCYTES
 MELANIN
• Light brown to black coloured pigment.
• Normally present in skin, eye, leptomeninges,adrenal
medulla,nerve Cells of substantia nigra and locus ceruleus.
• In skin, melanin is formed in melanocytes- large clear cells
in the basal layer of epidermis.

• Melanins are produced from tyrosine by the action of

tyrosinase enzyme.
• Tyrosinase converts Tyrosine to DOPA which is then
converted to an intermediate pigment which polymerizes to
Melanin.

• Melanins are divided into EUMELANINS(black or brown

pigments, insoluble in most solvents) &
PHAEOMELANINS (red to yellow, & are solouble in dilute
alkali solution.)
 ABNORMALITIES IN PIGMENTATION

• Generalised hyperpigmentation - is seen in

• Addison’s disease(due to incr. ACTH)
• Ch. Arsenic poisoning(rain drop pigmentation)
• Ch. Malnutrition
• Cachexia
• Felty’s syndrome
• Thyrotoxicosis
• Prolonged treatment with chlorpromazine(affect
lens,cornea,conjunctiva and retina)
 FOCAL HYPERPIGMENTATION
• FRECKLES (EPHELIDES)
Small light brown macules on the skin.
There is increased amt of pigment in the basal layer but no increase in no. of
melanocytes.darken on exposure to sunlight.

• LENTIGO
Benign localized hyperplasia of melanocytes.
lentinges do not darken on eposure to sunlight.

• CAFÉ-AU-LAUT SPOTS -large pigmented patches, resemble freckles microscopically,

but there is mild increase in no. of melanocytes. Found in -
Neurofibromatosis & Albright’s syndrome.

• MELANOCYTIC NEVUS
Tan to brown, small, uniformly pigmented, macules or papules with well-defined rounded
borders. Certain types may transform to melanoma.
DIMINISHED PIGMENTATION
• GENERALIZED
OCULOCUTANEOUS ALBINISM -Recessive condition Present
at birth. Skin is milky white, hair is white & iris is blue-
grey.Solar keratosis, BCC & SCC may develop in later life. No.
of melanocytes is normal but melanosomes contain little or no
melanin.Two type
a.Tyrosinase negative-there is no
enzyme so virtually no melanin is produced.
b.Tyrosinase positive-here some
pigmentation may occur later in life in sun exposed area.
PHENYLKETONURIA - Phenylalanine that accumulates
probably competes for tyrosinase & inhibits melanin formation.

• FOCAL
VITILIGO -Sharply defined areas of depigmentation of skin.
Melanocytes are absent in the affected areas & no melanin is
present. An autoimmune basis is suspected.
 DEMONSTRATION OF MELANIN

• Melanins can reduce both silver & acid ferricyanide

solutions.
DEMONSTRATION:
Masson-Fontana Method : Black
Schmorl’s ferric-ferricyanide reduction test :Dark blue.
• Melanins can be bleached by strong oxidising agents & are
insoluble in most organic solvents.
Pemanganate(takes 1-4hrs), Chlorate, Peroxide(takes 1-2
days) can bleach melanin.
DEMONSTRSTION OF MELANIN (contd)

• Enzyme Method- Cells capable of forming

melanin can be demonstrated by the DOPA
method. The enz. Tyrosinase that is localized
within these cells will oxidize DOPA to form an
insoluble brown-black pigment.

• Fluorescence – Shows yellow fluorescence when

exposed to formalin sp. useful when
demonstrating amelanotic melanoma.
 PIGMENTS MIMMICKING TO
MELANIN

• OCHRONOSIS- A condition in which a

melanin like pigment is present in cartilages
of the ear,larynx,joints,etc.

• Occurs in Alkaptonuria.

• The pigment is formed from Homogentisic

acid.
 ENDOGENOUS FAT DERIVED PIGMENTS

Derived from oxidised & polymerized products of lipid.

LIPOFUSCIN-
• Composed of polymers of lipids & phospholipids complexed with proteins.
• Not injurious to cells but is the sign of free radical injury & lipid
peroxidation.
• Appears as a yellow-brown, finely granular intracytoplasmic perinuclear
pigment.
• Found in liver, heart(brown atrophy), seminal vesicles & nerve cells in old
age & severe wasting.
• Electron microscopy- granules are highly electron dense have membranous
structure.
• Staining methods: LONG ZIEHL-NEELSEN METHOD–Magenta.
SUDAN BLACK B Technique – Black.
H & E – Yellow brown.
OIL RED O – Brilliant red.
LIPOFUSCIN GRANULES
IN CARDIAC MYOCYTE
 ENDOGENOUS FAT DERIVED
PIGMENTS (contd)

• CEROID PIGMENT
Derived from undigested remnants of
heterophagocytosed material & is found in
macrophages & liver cells.
Fails to stain with the ferric –ferrocyanide
reaction.
 DUBIN-JOHNSON PIGMENT

• Found in liver of pts with Dubin – Johnson

syndrome.
• Defect is in hepatocellular excretion of
conjugated bilirubin due to defective
canalicular protein-MRP2.
• Chronic conj. hyperbilirubinemia.
• Chraterised by presence of coarsely
granular, brownish black intracellular
pigment in the centri lobular hepatocytes.
• The pigment resembles Lipofuscin.
 MYOGLOBIN
This pigment released from the musles.
Causes-strenuous exercise in healthy
people.
following sea snake bite
malignant hyperpyrexia
type v glyogenosis
The urine is dark port-wine colour.
Gives positive result as hemoglobin to
different chemical test.
 COPPER
 50% of ingested Cu – absorbed in
duodenum.
 Transported to portal circulation complexed
with albumin & histidine.
 Free Cu dissociates & taken up by
hepatocytes.
 Cu binds with apoceruloplasmin to form
ceruloplasmin which is secreted into blood.
 Circulating ceruloplasmin is degraded
within hepatic lysosomes & the released Cu
is excreted into bile.
 WILSON’S DISEASE
 Autosomal Recessive disorder.
 Mutation of ATP7B gene located on chr 13.
 Def. in ATP7B protein causes a decrease in
Cu transport into bile, impairs its
incorporation into ceruloplasmin & inhibits
ceruloplasmin secretion into the blood.
 Accumulation of toxic levels of Cu in organs
sp. liver, brain & eye.
 Cu causes toxic liver injury through
production of ROS.
MORPHOLOGICAL CHANGES
• LIVER- Fatty change,macrovesicular
steatosis, vacuolated nuclei, acute &
chronic hepatitis, rarely massive necrosis
& cirrhosis.

• BRAIN- Toxic injury sp. in putamen

which shows atrophy & cavitation.

• EYE - Cu is deposited in Descemet’s

membrane in cornea- KAYSER
FLEISCHER RINGS.
DEMONSTRATION OF COPPER

• RUBEANIC ACID METHOD – Cu appears

greenish black.

• MODIFIED RHODAMINE TECHNIQUE-

Cu appears red to orange red.
 CALCIUM
 Inorganic Ca salt normally found in bone and teeth.
 Free ionic Ca in the biood can not be demonstrated
 Abnormal deposition of Ca found in-
Tuberculosis
Infarction(gandy-gamma bodies)
Atheroma of blood vessels
Malakoplakia of bladder(michaelis-
gutman bodies)
 Here Ca mainly occur in phosphate and carbonate salts.
 Ca salts are usually monorefringent but Ca oxalate is
birerefringent.
 DEMONSTRATION OF CALCIUM

• H&E - Basophilic amorphous granular or clumped intra

or extra cellular deposit.

• Alizarin Red S Method -Reacts with calcium to form

Alizarin red S Calcium complex which gives orange red
colour.(pH 4.2)

• Von Kossa stain -Silver nitrate is used which reacts with

Ca carbonate & phosphate. Bright light reduces Ag salts to
Ag. Calcium component is blackened by silver deposition.
CALCIUM DEMONSTRATION
 URIC ACID
• Uric acid is a breakdown product of purine
metabolism. Small amount is obtained from food.
• Most of it is excreted by kidneys.
• Circulates in the blood in the form of monosodium
urate.
• Urate crystals give a negative birefringence under
polarizing microscope.
• High mono Na urate may lead to gout,characterised by
subcutaneous nodular deposits of urate crystals
synovitis and arthritis
renal disease and calculi
• The urate crystals show argentaffin properties.
 ARTEFACT PIGMENTS
• FORMALIN PIGMENT
Seen as brown deposit in tissues fixed in acidic formalin.
Usually seen in blood rich tissues like spleen,hemorrhagic lesions
and large blood vessels filled with blood.
Birefringent with polarized light.
Treatment with 10% ammonium hydroxide in 70% alcohol for 5-15
min. removes the pigment.

• MALARIA PIGMENT
Morphologically similar to Formalin pigment.
It is formed within RBCs that contain malarial parasite. May also
be present in phagocytic cells which have ingested the infected
RBCs.
Exhibits birefringence.
Removed by saturated.alcoholic picric acid.(Requires 12-24 hrs
treatment)
 ARTEFACT PIGMENTS
• MERCURY PIGMENT
Seen in tissues that have been fixed in mercury containing
fixative.
Usually monorefringent brownish-black extracellular crystals.
Treatment with lugol’s iodine and subsequent bleaching with a
weak Na thiosulfate solution remove the pigment.
• CHROMIC OXIDE
Fine yellow-brown pigment.
The pigment is monorefringent and extracellular.
Removed by treatment with 1% acid alcohol.

• STARCH
Introduced by talcum powder from gloves of surgeons,nurses or
pathologists.
PAS+ve, shows Maltese cross configuration under polarised
light.
EXOGENOUS PIGMENTS &MINERALS

 They gain access to the body by

 inhalation

 ingestion

 skin implantation

 The most common minerals seen in tissue

section are carbon,silica &asbestos.
 CARBON
 Commonly found in lungs & LN of tobacco
smokers,urban dwellers
 Black pigmentation of lung(anthracosis)is
seen in coal workers
 Can not be demonstrated with conventional
histological technique
 Confused with melanin- treatment with
bleaching agent can easily differentiate.
 SILICA
 Mine workers inhale a large amount of
silica,give rise to the disease silicosis.
 Can not be demonstrated by routine
histological stains.
 It is anisotropic when examined using
polarized light.
 ASBESTOS
 The most dangerous type is crocidolite.The
fibres are 5-100um long & 0.25-0.5um in
diameter.
 Asbestosis is the disease caused by it, where
it is collected in the alveoli at the periphery
of the lung.
 The asbestos body looks beaded,yellow-
brown,dumb bell shaped in lung section.
TATTO PIGMENT

Some minerals in the form of dye

complex introduced in our body during
tattoing.
This pigments mainly found in the skin
and adjacent lymph node.
If viewed using reflected light various
colours of the dye pigments can be seen.
 TAKE HOME MASSAGE
The pigments are substances occurring in living matters,they
differ in their origin,chemical constitution and biological
significance but grouped together as they all absorb electro
magnetic energy of 400-800nm.
Pigments may be endogenous,exogenous and artefact
pigment.But endogenous pigments are more important from
pathological point of veiw.
Most of the pigments are identified in tissue by their specific
staining property ,like Perl’s stain for hemosiderin,Modified
fouchet’s method for bile pigment etc.
Demonstration of these pigments in tissues aid to clinical
diagnosis,like hemosiderin in hemosiderosis,porphyrin
pigment in porphyria,uric acid pigment in gout etc.
 TAKE HOME MASSAGE
Lipofuscin is not injurious to health,it is the pigment of free
radical injury and aging process.
The exogenous and artefact pigment serve no physiologic
function.The exogenous pigment can not be demonstrated by
routine histological technique.
 Y OU
N K
THA