Abnormal insoluble yellow, brown or black pigment deposits that can be seen without staining, are

frequently encountered in tissues submitted for histologic processing. Pigmentation is the process by
which substances that absorb visible light and produce color are deposited in the body in normal or
abnormal situations. Pigments can occur in normal or in pathological conditions and can be classified
into three major categories: endogenous, exogenous and artefacts of fixation.

1. Endogenous pigments are produced within the tissue to serve a physiological function, or may be by-
products of normal metabolism. They are further subdivided into hematogenous or blood-derived
pigments (hemosiderin, hemoglobin, bile pigment), nonhematogenous (such as melanin, lipofuscin and
chromaffin), and endogenous minerals (such as iron, calcium and copper). Endogenous pigments
become pathologic when they are deposited in excessive quantity or found in abnormal locations.

2. Exogenous pigments- Exogenous pigments consist of foreign materials, usually minerals introduced to
the body thru air, food, medication and injections. Examples of exogenous pigments are tattoos,
asbestos, carbon, silica, iron and silver. Carbon is the most common exogenous pigment, appearing as
jet black pigments in lung sections and bronchial glands of chronic smokers. Iron may be present as an
endogenous pigment in the liver in case of iron overload, or may be an exogenous pigment in the case
of shrapnel wound.

3. Artifact pigments – usually lie on top of tissue instead of within the cell. They are produced in tissues
during processing and most commonly result from fixation. Formalin pigment occurs when tissue is fixed
in acidic formaldehyde solutions.

Lillie’s Method for Ferric and Ferrous Iron (Lillie 1976)

Sections: Paraffin 6 microns.

Solutions: Potassium Ferrocyanide
Solution: Potassium Ferrocyanide 0.4 gm Hydrochloric Acid
40 ml Prepare fresh, just before use.
Potassium Ferricyanide Solution:
Potassium Ferricyanide 0.4 gm
Hydrochloric Acid 40 ml. Prepare fresh, just before use.
Results: Ferric iron Dark Prussian blue
Ferrous iron Dark Turnbull’s blue
Background light Red

Perl's Prussian Blue Method for Hemosiderin (ferric iron) (Perls 1867; Suvarna 2013)
Fixation: Neutral Formalin or other fixatives except acid fixative and potassium dichromate
Sections: The method works well on all types of sections.
Solution: Acid ferrocyanide
Solution: 1% aqueous potassium ferrocyanide
20 ml 2% aqueous hydrochloric acid
20 ml Preferably freshly prepared just before use.
Results: Hemosiderin and ferric salts stain deep blue.
Other pigments retain their natural color.
Tissues and nuclei stain red (according to counterstain).

Gomori's Prussian blue Stain for Iron (Luna 1968)

Fixation: Alcohol or l 0% formalin
Sections: Paraffin sections cut at 6 microns
Solution: Nuclear Fast Red Dissolve 0.1 gm. nuclear fast red in 100 cc. of 5% aluminum sulfate with aid of
heat. Cool and filter. Add a grain of thymol as preservative. Keep well at room temperature.
Results: Iron pigments bright blue
Nuclei red
Cytoplasm pink to rose

Leuco patent blue V Stain for Hemoglobin (Dunn and Thompson 1946; Suvarna 2013)
Fixation: Formalin
Solutions: Stock Solution: 1% aqueous patent blue V (CI 42045) 25 ml Powdered zinc 2.5 gm Glacial
acetic acid 0.5 mL
Results: Hemoglobin peroxidase (RBCs and neutrophils) dark blue
Nuclei red
Note: 1. Fixation in excess of 36 hours may give rise to unreliable results.

Bile Pigments and Hematoidin

Removal of iron from the heme of hemoglobin due to degradation of red blood cells, results in the
formation of biliverdin, a green compound that is transported to the liver, where is it further reduced to
bilirubin that is orange in color. Bilirubin is a bile pigment that is excreted by the hepatocytes of the
liver, removed from circulation in the blood and secreted into the duodenum as a component of bile.
When bilirubin is not excreted by hepatocytes, jaundice or yellowing of the skin can occur. Bile pigments
contain both conjugated and unconjugated bilirubin, biliverdin and hematoidin, all of which are
chemically distinct and show different physical properties. In H&E stained section of the liver, bile if
present is seen as small yellow-brown globules, first in the liver cells or hepatocytes, and later in the bile
canaliculi.
Biliverdin and bilirubin are considered bile pigments. Bile pigments can vary in color from yellowish-
brown to green. Jaundice, a yellow discoloration of the skin, is caused by excess bile pigment in patients
with liver failure, hemolytic anemia, or when there is an obstruction in the flow of bile from the liver to
bilirubin. In the liver, bile pigments appear in hepatocytes as yellowbrown globules which is sometimes
difficult to distinguish from lipofuscins that are also commonly seen within the cells. Both pigments
appear yellow brown on H&E stained paraffin sections (the green color of biliverdin is often masked by
eosin), and can be positive with Schmorl's ferric ferricyanide reduction test. Lipofuscin is autofluorescent
while bile pigments are not.

The most commonly used method for demonstration of bile pigments is Hall’s modified Fouchet
technique in which the pigment is converted to the green color of biliverdin and blue cholecyanin by the
oxidative reaction of the ferric chloride in the presence of trichloroacetic acid (Fouchet’s reagent). The
tissue section is then counterstained with Van Gieson’s solution. Only bile and bile pigments in the liver
are detected using this special staining technique. It is applied to paraffin sections of formaldehyde-fixed
tissue. The Gmelin’s technique is used to demonstrate bile pigments in other locations outside the liver.

Modified Fouchet's Technique for Liver Bile Pigments (Hall 1960)

Fixation: Any fixative is suitable.
Sections: Any section is suitable.
Solutions: Fouchet's solution 25% aqueous trichloroacetic acid 36 ml 10% aqueous ferric chloride 4 ml
Freshly prepared before use.
Van Gieson’s stain Dissolve 100 mg. of acid fuchsin (Cl 42685) in l 00 ml. of saturated aqueous picric acid.
RESULTS:
Bile pigments emerald to blue
Muscle green yellow Collagen red

Lipofuscin (hemofuscin
Lipofuscin is a yellow-brown to reddish-brown pigment that is due to a slow and progressive oxidation of
lipids and lipoproteins. Lipofuscin is known as “wear and tear pigment” or “brown atrophy” that is
usually associated with aging. It represents the indigestible, lipid containing remnants of autophagic
vacuoles formed during the cellular aging or atrophy and is formed in long-lived metabolically active, but
not mitotically active, cells. It can be found in hepatocytes, cardiac muscle cells, adrenal cortex, brain,
spinal cord, bone marrow, kidney and other organs. Normal lipofuscin contains fatty acids that are
closely associated with protein. This association allows most of the pigment to remain in place during
passage through the solvents used in paraffin embedding. It can be stained with oil-soluble dyes in
frozen sections and with basic fuchsin in paraffin embedded tissue. It is often reacts with a variety of
histochemical and staining methods, including stains for lipids, the Schmorl’s ferric-ferricyanide
reduction technique, the Fontana Masson silver stain method, Gomori 's aldehyde fuchsin, methyl
green, and occasionally with the Periodic Acid Schiff (PAS)method. Some lipofuscin can be stained with
Sudan black B and carbol fuchsin in a modified Ziehl-Neelsen stain.

Gomori's Aldehyde Fuchsin Technique for Lipofuscin (Gomori 1950)

Fixation: Any fixative
Sections: Works well on all types of sections
Results: Lipofuscin purple
Background yellow
Melanin is an autogenous pigment (brown or black), normally found in the skin, eyes and in pigment-
bearing neurons within the brainstem, such as the locus ceruleus and the substantia nigra. It is formed
by melanocytes when tyrosine is converted to dihydroxyphenylalanine (DOPA) by tyrosinase, and then
into melanin, packaged into protein containing granules called melanosomes. It is soluble in strong
alkali, and does not react with iron or fat stain. Pathological deposition of melanin occurs in benign
lesions such as a nevus or "mole", or in malignant tumors such as melanoma. A high percentage of
amelanotic melanomas can be identified with the monoclonal antibodies HMB-45, Melan A, and NSE.
The commonly used Fontana-Masson ("melanin stain") method relies upon the melanin granules to
reduce ammoniacal silver nitrate (but argentaffin, chromaffin, and some lipochrome pigments also will
stain black as well). Schmorl's method uses the reducing properties of melanin to stain granules blue-
green.
The most specific method of all is an enzyme histochemical method called DOPA-oxidase. It requires
frozen sections for best results, but paraffin sections of well-fixed tissues may be used. The stain works
because the DOPA substrate is acted upon by DOPA-oxidase in the melanin-producing cells to produce a
brownish black deposit. Two methods are commonly used: The MassonFontana silver method and
Schmorl’s ferric ferricyanide reaction.

Substances, such as melanin, which have the ability to bind and reduce silver without the use of a
separate reducing agent are said to be argentaffin, which means possessing the ability to reduce silver
without the aid of light or a reducing agent. The Fontana-Masson method is an argentaffin procedure
Chromaffin cells, also known as pheochromocytes, are neuro-endocrine cells found mostly in the
medulla of the adrenal glands. They release catecholamines: 80% of Epinephrine (Adrenaline) and 20%
of Norepinephrine (Noradrenaline) into systemic circulation. They are named as such because they can
be visualized by staining with chromium salts. Chromium salts oxidize and polymerize catecholamines to
form a brown color, most strongly in the cells secreting noradrenaline. They can be demonstrated by
Schmorls' staining solution that contains ferric chloride and potassium ferricyanide. However, because
other pigments give a positive reaction with Schmorl’s technique, a melanin bleach procedure with
potassium permanganate and oxalic acid should also be done.

Melanin is not extracted by acid treatments that remove formalin pigment. They are resistant to fat
solvents such as acetone but are soluble, to varying degrees, with hydrogen peroxidase, potassium
permanganate and chromic acid. In cases when melanin pigment obscures cellular detail, melanin
pigment can be bleached with potassium permanganate and oxalic acid solutions or 12-24 hours.
Melanin bleaching can be performed on free floating frozen or paraffin sections. The darker the melanin
pigment the longer the bleach will take to decolorize the pigment.

Masson-Fontana Method for Melanin (Masson 1928; Lillie 1965) Certain tissue components are
argentaffin; that is, they possess the ability to bind silver from a solution and to reduce it to visible
metallic silver without the need for a separate reducing agent. A 10% neutral buffered formalin can be
used as a fixative. Avoid alcohol because it dissolves argentaffin granules. Melanin has the ability to
reduce solutions of ammoniacal silver nitrate to metallic silver.
Fixation: Formalin
Sections: Standard paraffin sections
Results: Melanin black
Argentaffin granules black
Nuclei red