pigments in health and disease

1. Pigments with diagnostic
applications and
demonstration
Swarnendu Pal

2.  Introduction – definition
 Classification
 Endogenous pigments
 Artifact pigments
 Exogenous pigments and minerals
 Take home message

3. Pigments
 Pigments are defined as substances occurring
in living matter that absorb visible light
 May greatly differ in origin, chemical
constitution and biological significance.
 They can be either organic or inorganic

4. Classification
 Endogenous pigments
 Artifact pigments
 Exogenous pigments and minerals

5. ENDOGENOUS PIGMENTS

6. Endogenous pigments
 These substances are produced either within
tissues and serve a physiological function, or are
by-products of normal metabolic processes.
 They can be further subdivided into:
a. hematogenous (blood-derived) pigments
b. non-hematogenous pigments
c. endogenous minerals

7.  A) Hematogenous
This group contains the following blood-derived
pigments
hemosiderins
hemoglobin
bile pigments
porphyrins

8. Hemosiderin
 Hemoglobin-derived, golden yellow to brown,
granular or crystalline pigment normally appear
intracellularly.
 iron is in the form of ferric hydroxide that is bound
to a protein framework apoferritin, to form ferritin
micelles
 Hemosiderin pigment represents aggregates of
ferritin micelles

9. Hemosiderin
 Normal conditions - small
amounts in the
mononuclear phagocytes
of the bone marrow,
spleen, and liver,
 Local or systemic
excesses of iron 
hemosiderin accumulate
within cells.

10. Significance
 Increased hemosiderin deposition seen in
Hemorrhage in tissues - organizing hematomas,
hemorrhagic infarcts, fractures
systemic overload of iron - hemosiderosis.
The main causes of hemosiderosis are
1. increased absorption of dietary iron due to an
inborn error of metabolism called
hemochromatosis
2. Hemolytic anemias,
3. Repeated blood transfusions

11. Localised Hemosiderin Deposits
 Localized deposits in
dermatofibromas
 Pulmonary
hemosiderosis: seen in
mitral stenosis & left
ventricular failure –
‘heart failure cells’.

12. Demonstration of hemosiderin
 Perls’ Prussian blue reaction
 Principle :
Detection of ferric Fe3+ iron in tissues. Strongly
bound iron as hemoglobin will not react.
Sections treated with acidic solution of potassium
ferrocyanide and ferric iron present unmasks as
ferric hydroxide reacts to form an insoluble bright
blue pigment ferric ferrocyanide known as prussian
blue
 3 K4Fe(CN)6 + 4Fe3+  Fe4[Fe(CN)6]3 + 12K+

13. Perls’ Prussian blue reaction
 Fixative : Alcohol/ 10%
neutral buffered
formalin. Avoid the use
of acid fixatives
 Control : postmortem
lung tissue (heart failure
cells)
 Sections - Works well on
all types of section
Results
 Ferric iron - blue
 Nuclei - red

14. Turnbull’s blue stain for ferrous Fe2+
iron (Tirmann Schmetzer’s)
 Principle :
◦ The method is very similar to Perls' Prussian blue, but
uses potassium ferricyanide instead of ferrocyanide.
◦ The ferrous iron reacts with the potassium
ferricyanide to form ferrous ferricyanide blue
compound known as Turnbull's blue.
Results
 Ferrous salts and ferric salts converted by treatment with
ammonium sulphide - Deep blue
 Nuclei - Red

15. Other methods
 Lillie’s method for ferric and ferrous iron
◦ Ferric iron dark Prussian blue
◦ Ferrous iron dark Turnbull’s blue
◦ Nuclei red
 Hukill and Putt’s method for ferrous and ferric
iron
◦ Ferrous iron red
◦ Nuclei blue

16. Hemoglobin
 Basic conjugated protein
 Composed of a colorless protein, globin, and a red
pigmented component, heme.
 Histochemical demonstration of the ferrous iron is
possible only if the close binding in the heme
molecules is cleaved

17. Hemoglobin
 Need to demonstrate the pigment may arise in
1.casts in the lumen of renal tubules in cases of
hemoglobinuria
2.active glomerulonephritis
 Methods of demonstration :
A. Demonstration of enzyme Hb peroxidase by
benzidine – nitroprusside method (carcinogenic)

18. Methods of demonstration
Hemoglobin
 B. Leuco patent blue V
method for hemoglobin
◦ Results
 Hemoglobin peroxidase
(RBCs and neutrophils) -
dark blue
 Nuclei – red
 C. Tinctorial methods
◦ the amido black
technique
◦ the kiton red-almond
green technique

19. Bile pigments
 Bile pigments are both conjugated and unconjugated
bilirubin, biliverdin, and hematoidin
 Excess of bile pigments,
bile duct obstruction
Abnormality of bilirubin biliverdin metabolism
(congenital enzyme disorders)
Extensive liver cell death/ degeneration

20. Bile pigments
 Bile pigments appear as
yellow-brown globules.
 Confused with lipofuscin.
 (need to identify bile
pigments mainly in the
hp examination of the
liver)

21. Bile Pigments vs Lipofuscin
 H/E : Both appear yellow-brown in H&E-stained
paraffin sections
 lightly counterstained with a suitable Mayers
hematoxylin
 Under polarized light Bile pigments are not
autofluorescent and fail to rotate the plane of
polarized light (monorefringent)
 lipofuscin is autofluorescent

22. Demonstration :
A. Modified Fouchet technique-
 Principle :
bilirubin converted to green coloured biliverdin in
an acidic medium.
This oxidation is accomplished by ferric chloride in
trichloroacetic acid medium (Fouchet reagent)
(freshly prepared)
 Fixation - Any fixative appears suitable.

23.  Results :
Bile pigments -
emerald to blue-
green
Muscle - yellow
Collagen - red

24. Bile pigments
 B. Gmelin technique-
Treatment with concentrated nitric acid
changing colour spectrum: yellow-green-blue-
purple-red.
Less specificity and unreliable. Test should be
repeated at least three times before accepting
the result as negative.

25. Porphyrin pigments
 Normally occur in tissues in only small amounts.
 Precursors of the heme portion of hemoglobin
 Appears as a dense dark brown pigment
 In fresh frozen sections exhibits a brilliant red
fluorescence that rapidly fades with exposure to
ultraviolet light.

26. Porphyrin pigments
 The pigment, when
seen in paraffin
sections and viewed
using polarized light,
appears bright red in
color with a
centrally located,
dark Maltese cross.

27. NON-HEMATOGENOUS
PIGMENTS

28. Non-hematogenous endogenous
pigments
This group contains the following:
 melanins
 lipofuscins
 chromaffin
 pseudomelanosis (melanosis coli)
 Dubin-Johnson pigment
 ceroid-type lipofuscins
 Hamazaki-Weisenberg bodies

29. Melanin
 Light brown to black
pigment
 Normally found in skin,
eye, substantia nigra of
the brain, and hair
follicles
 Pathological conditions -
found in benign nevus
cell tumors and
malignant melanomas.

30. Melanin
 Tyrosine  DOPA  Intermediate pigment 
Melanin
 melanins are bound to proteins, and these
complexes are localized in the cytoplasm of
cells within so-called ‘melanin granules’

31. Methods of demonstration of
Melanin
The most reliable of these are:
1. Reducing methods such as the Masson-Fontana silver
technique and Schmorl’s ferric ferricyanide reduction
test.
2. Enzyme methods (e.g. DOPA reaction).
3. Solubility and bleaching characteristics.
4. Fluorescent methods.
5. Immunohistochemistry (melanin activation
antigens).

32. Melanin
 1. Reducing methods for melanin
Melanin is a powerful reducing agent and this property is
used to demonstrate melanin in two ways:
A. The reduction of ammoniacal silver solutions to form
metallic silver without the use of an extraneous
reducer is known as the argentaffin reaction
 Masson-Fontana method for melanin and churukian
ammoniacal silver method for argentaffin and
melanin
 Melanin, argentaffin, chromaffin and lipofuscins
black
Nuclei - red

33. Melanin
 B. Melanin will reduce ferricyanide to ferrocyanide
with the production of Prussian blue in the presence
of ferric salts (Schmorl reaction)
Melanin, argentaffin, chromaffin and lipofuscins –
dark blue
Nuclei - red

34. Schmorl’s methodChurukian’s method

35.  2. Enzyme methods
 Cells that are capable of producing melanin can be
demonstrated by the DOPA(dihydroxyphenylalanine)
method.
 Enzyme tyrosinase localized within cells will oxidize
DOPA to form an insoluble brown-black pigment
 DOPA oxidase(tyrosinase) – brown, nuclei - red

36.  3. Solubility and bleaching methods
 Melanins are insoluble in most organic solvents (due
to tight bond with protein component)
 Use of strong oxidizing agents, such as
permanganate, chlorate, chromic acid, peroxide,
and peracetic acid,  bleach melanin
 Method of choice :
Peracetic acid,
Treatment with 0.25% potassium permanganate
followed by 2% oxalic acid

37.  4. Formalin-induced fluorescence (FIF)
 Aromatic amines such as 5-HT, dopamine,
epinephrine (adrenaline), norepinephrine
(noradrenaline), and histamine, when exposed to
formaldehyde, show a yellow primary fluorescence
 Useful in amelanotic melanoma

38.  5. Immunohistochemistry
 Ideally used are S100, HMB 45 and PGP 9.5
 S100 protein antigen gold standard antibody remains
expressed in cells derived from the neural crest
(so not specific for melanocytes alone)
 HMB 45 demonstrates melanosome formation and
melanocytic differentiation (Not a melanoma Ag)

39. Ochronosis
 Melanin-like pigment, present in cartilages of
ear, larynx, trachea, joints etc.
 Formed from homogentisic acid, in alkaptonuria.

40. Lipofuscins
 Yellow to red-brown
pigments
 produced by an
oxidation process of
lipids and lipoprotein
 Hepatocytes, Cardiac
muscle cells, adrenal
cortex, Testis, ovary,
spinal cord, and
ganglia, edge of a
cerebral hemorrhage
or infarct, involuntary
muscle, cervix, and
kidney

41. Methods of demonstration
The most common and useful being:
 Periodic acid-Schiff method
 Schmorl’s ferric-ferricyanide reduction test
 Long Ziehl-Neelsen method
 Sudan black B method
 Gomori’s aldehyde fuchsin technique
 Masson-Fontana silver method
 Basophilia, using methyl green
 Churukian’s silver method
 Lillie’s Nile blue sulfate method

42. Gomori’s aldehyde fuchsin.Lipofuscin stained purple

43. Chromaffin
 This pigment is normally found in the cells of the
adrenal medulla as dark brown, granular material.
 It may occur in tumors of the adrenal medulla -
pheochromocytomas.
 Demonstrated by Schmorl’s reaction, Lillie’s Nile blue
A, the Masson-Fontana, Churukian’s microwave
ammoniacal silver method, and the periodic acid-
Schiff (PAS) technique

44. Pseudomelanosis pigment
(melanosis coli)
 Seen in macrophages in
the lamina propria of the
large intestine and
appendix
 Endogenous lipopigment
 Associated with purgative
use
 Stains similar with those
of lipofuscin

45. Dubin-Johnson pigment
 Found in liver of patients
with Dubin-Johnson
syndrome
 histochemically similar to
lipofuscin
 Presence of a brownish-
black, granular,
intracellular pigment
situated in the
centrilobular hepatocytes

46. Ceroid type lipofuscin
 found in hepatic
cirrhosis
 lipofuscin at an early
stage of oxidation.

47. Hamazaki-Weisenberg bodies
 Small, yellow-brown,
spindle-shaped structures
found in the sinuses of LN
 significance unknown.
 present in patients of
sarcoidosis
 Histochemically they are
similar to lipofuscin may
represent lysosomal
residual bodies

48. ENDOGENOUS
MINERALS

49. Calcium
Abnormal depositions of calcium found in
 necrotic areas of tissue associated with
tuberculosis,
 Infarction (Gandy-Gamna bodies),
 atheroma in blood vessels,
 malakoplakia of the bladder (Michaelis-Gutman
bodies)

50. Calcium
Gamma - Gandy bodies Michaelis-Gutman

51. Method of demonstration
H/E stain Alizarin red S method for calcium

52. von Kossa’s silver nitrate method for calcium

53. Copper
 Associated with Wilson’s
disease
 Demonstrated by :
 Rubeanic acid method
for copper
 Modified rhodanine
technique

54. Uric acid and urates
 Breakdown product of purine metabolism
 Urate crystals give a negative birefringence under
polarizing microscope.
 May lead to gout, characterised by,
i) subcutaneous nodular deposits of urate crystals,
ii) synovitis and arthritis
iii) renal disease and calculi

55. Uric acid and urates
Negative birefringence
Gouty tophi H/E stain

56. ARTIFACT PIGMENTS

57. Artifact pigments
This group of pigments comprises:
 formalin
 malaria
 schistosome
 mercury
 chromic oxide
 starch

58. Formalin pigments
 brown or brown-black deposit in tissues fixed in
acidic formalin.
 The deposit is usually present in blood-rich tissues
such as spleen, hemorrhagic lesions, and large
blood vessels filled with blood
 use of buffered neutral formalin will help to
minimizes the problem

59. Removal of formalin pigments
 Treating unstained
tissue sections with
saturated alcoholic
picric acid
 Treatment with 10%
ammonium hydroxide
in 70% alcohol for 5–15
minutes

60. Malarial pigment
 Morphologically
similar to Formalin
pigment.
 formed within RBCs
that contain malarial
parasite.
 Removed by sat.
alcoholic picric acid.
Requires 12-24 hrs
treatment.

61.  Schistosome pigment
This pigment is occasionally seen in tissue
sections where infestation with Schistosoma is
present.
 Mercury Pigment
 Brownish-black,extracellular crystal tissues that
have been fixed in mercury-containing fixatives.
 Removed by treatment with Iodine soln

62.  Chromic oxide
 tissues that have been fixed in chromic acid or
dichromate-containing fixatives
 Removed by treatment with 1% acid alcohol
 Starch
 This pigment is introduced by powder from the
gloves
 PAS and GMS positive, when polarized, will
produce a Maltese cross configuration

63. EXOGENOUS PIGMENTS
& MINERALS

64. Carbon
 Commonly inhaled, ingested
or implanted in skin following
industrial exposure.
 lungs and LNs of coal
workers- anthracosis
 In skin tissues confused with
melanin deposition, but
treatment with bleaching
agents - carbon unaffected,
melanin - colour disappear.

65. Silica
 causes disease silicosis
 presents with
progressive
pulmonary fibrosis
 histochemically inert,
but birefringent.

66. Asbestos
 Asbestos: birefringent fibres
of magnesium silicate.
 Collect in alveoli, at
periphery of lung.
 Asbestos body-
beaded,yellow-brown,
barbell-shaped with
proteinaceous coat
containing hemeosiderin.
- demonstrated by prusian
blue reaction.

67. Tattoo pigments
found in the skin and
adjacent lymph node
of various colours of
the dye pigments
seen.

68. Others due to industrial exposure
 Lead
 Silver
 Beryllium and aluminum

69. Take home message
 Demonstration of pigments in tissues is an aid to
diagnosis
 Pigments may present itself in tissue sections with
variety of morphology.
 It is advisable to note a pigment’s morphology, tissue
site & relevant clinical data, to carry out various
special stains for confirmation

70. References
 Bancroft’s Theory and Practice of Histological
Techniques 6th edition, 2008
 Robbins and Cotran Pathologic Basis of Disease 9th
edition,
 Handbook of Histopathological and Histochemical
Techniques, C. F. A. CULLING 3rd edition
 Surgical Pathology- 12th ed.- Rosai & Ackerman

71. Thank
You