Histopathology topic deal with pigments demonstration

1. Pigments
Mr.Mahmoud Ibrahim Osman
MSc in Histopathology

2. Pigments
substances occurring in living matter ,
that absorb visible light , they can be
either organic or inorganic that remain
insoluble in most solvents.

3. Pigment can be classified under the following
heading:
1. Endogenous pigments
2. Exogenous pigments
3. Artifact pigments

4. Endogenous pigments
• These substances are produced either
within tissues and serve a physiological
function or product of normal metabolic
.

5. • They can be subdivided into:
1. Hematogenous (blood derived pigments)
2. Non- hematogenous pigments
3. Endogenous minerals

6. Hematogenous pigments
• This group contains the following
blood-derived pigments:
1. Hemosiderin
2. Hemoglobin

7. 1. Bile pigments
2. Porphyrins

8. Hemosiderin
• Yellow to brown granules ( intracellularly )
• They contain iron in form of ferric
hydroxide bound to a protein frame work.
Beside hemosiderin iron also occur in :
1 .Hemoglobin 60%

9. 2 .Myoglobin
3 .Certain enzymes e g cytochrome oxide and
peroxides

10. Iron Metabolism
• Dietary iron is absorbed in the small
intestine and attached to a protein
molecule for transfer to the sites in the
body where it is to be utilized or stored.

11. • Approximately 30% is stored within the
reticuloendothelial system, especially the
bone marrow. The bone marrow is also
the main site of iron utilization in the
body.

12. • Iron loss:
1. Epithelial desquamation
2. Hair loss
3. Sweating

13. 1. Hemorrhages
A-chronic bleeding (e.g. a peptic ulcer,
bowel neoplasia)
B-menstruation.

14. • In iron deficiency, the iron stores in the
bone marrow become depleted,
insufficient hemoglobin is produced
because of the lack of iron, and anemia
develops.

15. • Iron excess is a much less common condition,
because under normal conditions the
intestine will not absorb iron from the diet
when there is already a surplus within the
body.

16. • Iron overlapping :
1. iron injection
2. Blood transfusion
3. Impair control of iron absorption in
small intestine

17. • Hemosidrosis : deposition of excessive
amount of hemosiderin (due to iron
injection or blood transfusion ) in organs
mainly RES.

18. • Hemochromatosis :large quantities of
hemosiderin ( due to impaired control of
iron absorption ) deposited in many
organs interfering with organs structure
and function.

19. Demonstration of hemosiderin
• In unfixed tissue hemosiderin is
insoluble in alkalis ,but soluble in acid
solution
• Fixatives contain acids can remove
hemosiderin granules

20. • Iron occur in tissue in ferric salt (mainly
) and in ferrous salts .

21. Perl's Prussian Blue for ferric iron
• Treatment with diluted hydrochloric
acid will result in the unmasking of
ferric iron from proteins then ferric
iron then react with a diluted
potassium

22. • ferrocyanide to produce an insoluble
blue compound (ferric ferrocyanide –
prussian blue ).
• Avoid use of acid fixatives and
chromate fixatives.

23. Perls Prussian Blue- Hemosidrosis Liver -
Iron stain

24. Turnballs` blue for ferrous salts
• Interaction of ferrous salts with
potassium ferricyanide is result in
ferrous ferricyanide (insoluble blue
compound)

25. Quinkes` and Schmeltzer reaction
• Used to demonstrate both ferrous ferric
iron Quinke test is depend upon the
reduction of ferric iron into ferrous iron
by ammonium sulphide .

26. • Forming green –black ferrous sulphide
which is converted into Turn blue by
Schmeltzer reaction .
• Lillie’s method for ferric and ferrous
iron

27. • Hukill and Putt’s method for ferrous and
ferric iron .

28. Bile pigment
• Red blood cells are broken down in RES
after 120 days , hemoglobin convert into
heme and globin , the tetra pyrrole ring
of heme is opened out and iron is
removed result in biliverdin .

29. • This residue is formed in the phagocytic
cells in RES ,biliverdin is transported to the
liver . Where it is reduced to bilirubin ,
the bilirubin become soluble in water
after conjugation with glucuronic acid.

30. • Then the conjugated bilirubin pass into
bile canaliculi then gall bladder then to
the duodenum.

31. • In H&E stained section of liver (bile if
present ) , seen in hepatocytes as yellow
– brown granules and must be
distinguished from lipofuscins .

32. • In bile canaliculi appear larger (round end
rods ) indicate obstructive jaundice .
• Bile is also seen in H&E-stained
sections in the gallbladder where it can
appear as amorphous, yellow-brown masses .

33. • adherent to the mucosa or included as
yellow-brown globules within the epithelial-
lined Aschoff-Rokitansky sinuses in the
gallbladder. Bile is also present, together with
cholesterol, in gallstones.

34. Hematoidin pigment
• Extra cellular yellow-brown crystals
and amorphous masses .
• Heme undergo a chemical change (its`
nature)
• Found in old hemorrhagic areas e g ; old
splenic infarcts , brain.

35. Demonstration of bile and
hematoidin
• Fouchet technique : it is a routine method
-simple –quick , bile pigment is
converted to green / blue color by the
oxidative action of ferric chloride in the
presence of tri chloracetic acid.

36. Gmelin technique
• This technique is the only method that
shows an identical result with liver bile,
gallbladder bile, and hematoidin. This
method gives impermanent results.

37. • Deparaffinized sections of tissue containing bile
pigments are treated with nitric acid, and a
changing color spectrum is produced.

38. • A popular modification of this technique
is that of Lillie and Pizzolatto in which
bromine in carbon tetrachloride is used
as an oxidant.

39. Hemoglobin
• Hemoglobin is a basic conjugated
protein that is responsible for the
transportation of oxygen and
carbon dioxide within the bloodstream.

40. • It is composed of a colorless protein,
globin, and a red pigmented component,
heme.

41. Demonstration of hemoglobin
• Two types of demonstration methods can
be used to stain hemoglobin in tissue
sections. The first demonstrates the
enzyme (hemoglobin peroxidase )and
Patent blue method.

42. A section of kidney from a patient with
hemoglobinuria stained for hemoglobin with leuco patent blue
V. Hemoglobin is stained blue.

43. Porphyrin pigment
• These substances normally occur in
tissues in only small amounts. They are
considered to be precursors of the heme
portion of hemoglobin. The porphyrin
are rare pathological conditions.

44. Non -Hematogenous Endogenous
pigments
• These are include:
1. Melanin
2. Lipofuscins
3. Chromaffin
4. Pseudo melanosis (melanosis coli)

45. 4. Dubin-Johnson pigment
5. Ceroid-type lipofuscins
6. Hamazaki-Weisenberg bodies

46. Melanin's pigment
• Group of pigments varies from light brown to
black
• Produced from tyrosine by the action of an
enzyme tyrosinase (DOPA oxidase ). this
enzyme acts on tyrosine to produce DOPA
(dihydroxy phenylalanine ).

47. • and by the same enzyme DOPA convert
to melanin.
• Tyrosine (amino acid) tyrosinase enzyme=DOPA(3,4
dihydoxyphenylalanine)
• DPOA Tyrosinase enzyme===Melanin

48. Melanin Demonstration
1. Reducing methods
2. Enzyme method
3. Solubility and bleaching
4. Fluorescent method
5. Immunocytochemistry

49. Reducing methods
• Melanin is a powerful reducing agent
and this property is used to demonstrate
melanin in two ways.

50. • 1. The reduction of ammoniacal silver
solutions to form metallic silver without
the use of an extraneous reducer is
known as the argentaffin reaction.
Masson’s Fontana method.

51. • 2. Melanin will reduce ferricyanide to
ferrocyanide with the production of
Prussian blue in the presence of ferric
salts the Schmorl reaction.

52. Masson Fontana method for
melanin

53. Melanin pigment in cells of malignant melanoma ,
Fontana-Masson stain.

54. Enzyme method
• Cells that are capable of producing
melanin can be demonstrated by adding
DOPA , the enzyme that localized in
these cells will oxidize DOPA to form
brown pigment.

55. • The best results are obtained when using
post fixed cryostat sections.

56. Solubility and Bleaching
• Melanin are insoluble in most solvents ,
but can be bleached (decolorize ) by use
strong oxidizing agents e g
permanganate , chlorate , chromic acid ,
peroxide , peracetic acid .

57. Fluorescent method( Formalin-induced
fluorescence (FIF)
• Certain aromatic amines such as 5-
hydroxy tryptamine, dopamine,
epinephrine when exposed to
formaldehyde, show a yellow primary
fluorescence.

58. • This is particularly useful when
demonstrating amelanotic melanoma,
because these tumors can be difficult to
diagnose using conventional methods
due to their lack of pigment.

59. Immunohistochemistry
• Use of melanocyte selective antibodies to
highlight melanocytic lesions. The large
majority of these antibodies recognize an
antigen associated with melanocyte
activation, gp100 (HMB 45) or Mart-1
(Melan A).

60. Lipofuscin pigment
• Yellow –brown to reddish pigment
produced by an oxidation process of
lipids and lipoprotein , occur in
hepatocytes , cardiac muscles , adrenal
cortex , testis , ovary , neurons
• Lipofuscin is auto fluorescent

61. • The oxidation process occurs slowly and
progressively, and therefore the pigments
exhibit variable staining reactions, different
colors, and variation in shape and size,
which appears to be dependent upon their
situation

62. • Lipofuscins can be found in the following
sites:
• Hepatocytes.
• Cardiac muscles particularly around the
nucleus.
• Inner reticular layer of the normal adrenal
cortex.
• Testis.
• Ovary.

63. • Cytoplasmic inclusions in the neurons of the
brain.
• Some lipid storage disorders e.g. Batten's
disease.
• Other tissues such as bone marrow,
involuntary muscles, cervix and kidney.

64. Demonstration of Lipofuscin
1. Periodic acid –Schiff method
2. Schomrl`s reducing method
3. Long Z-N method
4. Sudan black B method
5. Masson –Fontana silver technique
6. Basophilia – using methyl green

65. • 7. Churukian’s silver method
• 8.Lillie’s Nile blue sulfate method
• 9. Gomori’s aldehyde fuchsin technique

66. A section of liver stained with Gomori’s
aldehyde fuchsin .Lipofuscin is stained
purple.

67. 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).

68. • Fixation in formalin is not
recommended, and fixatives
containing alcohol, mercury
bichloride, or acetic acid should be
avoided.

69. • Orth’s or other dichromate-containing
fixatives are recommended.

70. Demonstration of Chromaffin
• Chromaffin may be 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

71. Pseudomelanosis pigment (melanosis
coli)
• This pigment is sometimes seen in
macrophages in the lamina propria of the
large intestine and appendix.
• .It appears to be strongly associated with
anthraquinone purgatives (‘cascara sagrada’).

72. • Pseudomelanosis will, in general, stain
with those methods that are used to
demonstrate lipofuscin, such as Masson-
Fontana and Schmorl.

73. Dubin-Johnson pigment
• This pigment is found in the liver of
patients with Dubin-Johnson syndrome
and is due to defective canalicular
transport of bilirubin.

74. It is characterized by the presence of a
brownish-black, granular, intracellular
pigment histochemically it is similar to
lipofuscin, though there are
ultrastructural differences.

75. Ceroid-type lipofuscins
• Different from lipofuscin because it failed to
stain with the ferric-ferrocyanide reaction.
States that ceroid is in fact a lipofuscin at an
early stage of oxidation. Further oxidation
would produce lipofuscin proper.

76. Hamazaki-Weisenberg bodies
• These small, yellow-brown, spindle-
shaped structures are found mainly in
the sinuses of lymph nodes, either lying
free or as cytoplasmic inclusions and
their significance is unknown.

77. • Histochemically they are similar to
lipofuscin, and at ultrastructural level
have an appearance that suggests that
they are probably giant lysosomal
residual bodies

78. Endogenous minerals
• Calcium:
• Insoluble inorganic calcium salts are a
normal constituent of bones and teeth.
• Abnormal depositions of calcium can be
found in necrotic areas of tissue associated
with tuberculosis,

79. • infarction(Gandy-Gamna bodies),
atheroma in blood vessels, and
malakoplakia of the bladder (Michaelis-
Gutman bodies)

80. • Calcium usually stains purple blue with H&E.
• The classic method is von Kossa which uses
silver nitrate, is generally preferred for
routine demonstration.
• Alizarin red S,

81. Section of kidney stain with von
kossa

82. A section of kidney with calcium oxalate
crystals as seen with polarization
microscopy

83. Copper
• Many enzymes in the body would fail to
function without the presence of copper.
although copper deficiency is extremely rare.
Copper accumulation is associated with
Wilson’s disease.

84. • The rhodanine method has also been
used to demonstrate copper and copper-
associated protein (CAP).

85. A section of fetal liver of the third
trimester stained with Lindquist’s method
for copper. Copper is stained red to
orange-red

86. Uric acid and urates
• Uric acid is a breakdown product of the
body’s purine (nucleic acid) metabolism,
although a small proportion is obtained
from the diet.

87. • The uric acid circulating in the blood is in the
form of monosodium urate, which in patients
with gout may be high, forming a
supersaturated solution. These high levels
may result in urate depositions,

88. • Which are water soluble in tissues, causing
subcutaneous nodular deposits of urate
crystals (known as ‘tophi’).
• synovitis and arthritis
• renal disease and calculi

89. • Another condition that occasionally can
mimic Gout is known as pseudogout or
chondrocalcinosis. This results in
calcium pyrophosphate crystals being
deposited in joint cartilage.

90. • The cause of this deposition is unknown
and is more common in the elderly,
affecting mainly the large joints, such as
the knee.

91. • To aid the diagnosis, a polarizing
microscope is used.
• Whilst pyrophosphate crystals exhibit a
positive birefringence urate crystals
show a negative birefringence.

92. • Urates can be extracted by saturated
aqueous lithium carbonate solution
while pyrophosphate crystals are
unaffected.

93. Artifact pigments
• This group of pigments comprises:
1. Formalin pigment
2. Malaria pigment
3. Mercury pigment

94. 4. Chromic oxide pigment
5. Starch pigment
6. Stain precipitates
7. Schistosome pigment

95. Formalin pigment
• Brown to black (formaldehyde acid hematin ) ,
microcrystalline , extra cellular , birefringent
pigment. Deposit in tissues (blood rich
tissue such as spleen , hemorrhagic lesions
and large blood vessel) that have been fixed
in acid formalin .

96. • It is best avoided by the use of neutral
or buffered formalin
• Removal of formalin pigment:
1. Treating unstained section with
saturated alcoholic picric acid

97. 2. Alcoholic solution of both sodium and
potassium hydroxide (have effect on
staining )
3. Treatment with 10% ammonium
hydroxide in 70% alcohol

98. Malaria pigment
• Morphologically similar to formalin pigment
, but malaria pigment is intra cellular ,
formed within red blood cells that contain the
malaria parasite , also may present in
phagocytic cells which ingest infected red cell

99. • It is birefringence and can be removed with
alcoholic picric acid

100. Mercury pigment
• This pigment is seen in tissues that have
been fixed in mercury-containing
fixatives, although it is rarely seen in
tissue fixed in Heidenhain’s Susa.

101. Mercury pigment varies in its appearance but it
is usually seen as a brown-black, extracellular
crystal. Treatment of sections with iodine
solutions, such as Lugol’s iodine, is the classical
method of removing the pigment.

102. • Subsequent bleaching with a weak
sodium thiosulfate (hypo) solution
completes the treatment.

103. Schistosome pigment
• This pigment is occasionally seen in tissue
sections where infestation with Schistosoma
is ‘present’. The pigment, which tends to be
chunky, shows similar properties to those of
both formalin and malaria pigments.

104. Chromic oxide pigment
• Fine brown to black granules , occur after
dichromate fixation (e g zenker )
• Can be removed by several washing in water
after fixation and from section by 1% acid
alcohol
• It is monorefringence and extra cellular

105. Starch pigment
• Introduced by powder from the gloves
• It is PAS positive

106. • Stain precipitates :
• Amorphous and granular and sometime
crystalline
• These follow over oxidation or in evaporation
of saturated alcoholic solution

107. Exognous Pigments
• Although often listed as being exogenous
pigments, the majority of the following
substances are, in fact, colorless.
• Some of these substances are inert and
unreactive.

108. Exognous Pigments entering the organism via
 traumatic lesions
 gastrointestinal tract
 respiratory tract

109. traumatic origin
mechanic instilation of inert dyes into
the deep dermis.

110. Tattoo Pigment
• This is associated with skin and any adjacent
lymphoid areas. If viewed using reflected
light, the various colors of the dye pigments
used to create the tattoo can be see.

111. A section of skin demonstrating exogenous tattoo
pigment in a tattoo granuloma stained with hematoxylin
and eosin

112. Amalgam tattoo
• Brown-black areas of pigmentation in the
mouth may result from traumatic
introduction of mercury and silver from
dental amalgam during dental procedures.

113. Exogenous Pigmentation through Airways
• Carbon:
• This exogenous substance is the most
commonly seen mineral in tissues and is
easily recognized in stained tissue sections.

114. • Commonly found in the lung and adjacent
lymph nodes of urban dwellers and tobacco
smoker, the main sources of this material are
car exhausts.
• Carbon particles will be seen in
macrophage

115. • Heavy black pigmentation of the lung
(anthracosis)may be seen as a result of
massive deposition of carbonaceous matter in
coal workers.

116. • The lung disorder known as coal
workers’ pneumoconiosis is caused by
the inhalation of silica, coal dust, and
many other particulates.

117. • The carbonaceous material is relatively inert
and fails to be demonstrated with
conventional histological stains and
histochemical methods. The site and nature of
the carbon deposits make identification
relatively easy.

118. Silica
• Silica is un reactive –it is birefringence when
examined using polarized light . Mine
workers can inhale large quantities of silica,
which can give rise to the disease silicosis.

119. • This disease may present as a progressive
pulmonary fibrotic condition which gives rise
to impaired lung capacity and in some cases
extreme disability. Silica is unreactive, and
is thus not demonstrated by histological
stains and histochemical methods.

120. Asbestos
• Special form of silica , cause pulmonary
disease , yellow – brown dumb-bell shape.
Asbestos has been used for many years as a
fire- resistant and insulating material.

121. Lead
• Lead in paint, batteries ,and gasoline has been
reduced by the various manufacturers. Cases
of lead poisoning are rare and are usually
diagnosed biochemically using the serum from
suspected cases.

122. • The most popular method is the rhodizonate
method

123. Beryllium and aluminum
• The same methods are used to demonstrate both
of these metal.
• Solochrome azurine method for beryllium and
• aluminum

124. Silver
• Silver is occasionally found in the skin of
silver workers as a result of industrial
exposure. Rhodanine method for silver