PIGMENTS STAINING

OUTLINE
1.Introduction
2. Endogenous pigment
2.1 Hematogenous
2.1.1 Hemosiderin
2.1.2 Hemoglobin
2.1.3 Bile pigment
2.1.4 Porphyrin pigments
2.2 Non hematogenous
2.2.1 Melanins
2.2.2 Lipofuscins
3. Artifacts pigment
4. Exogenous pigments
 INTRODUCTION
 In biology, pigments are defined as substances
occurring in living matter that absorb visible light
(electromagnetic energy within a narrow band that
lies approximately between 400 and 800 nm).
 various pigments may greatly differ in origin,
chemical constitution and biological significance.
 They can be either organic or inorganic compounds
that remain insoluble in most solvents.
 Pigments can be classified as endogenous,
exogenous and artifacts pigments
 ENDOENOUS 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:
1. Hematogenous
• This group contains the following blood-derived pigments:
A. Hemosiderins
 These pigments are seen as yellow to brown granules and normally appear
intracellularly.
 They contain iron in the form of ferric hydroxide that is bound to a protein framework
and is unmasked by various chemicals.
Demonstration of hemosiderin and iron
Hemosiderins can be demonstrated by the following methods;
-Perls’ Prussian blue reaction
-Lillie’s method
-Hukill and Putt’s method
 Perls’ Prussian blue reaction
 This method is considered to be the first classical histochemical reaction.
 Treatment with an acid ferrocyanide solution will result in the unmasking of ferric iron in the form of the
hydroxide, Fe(OH)3, by dilute hydrochloric acid.
 The ferric iron then reacts with a dilute potassium ferrocyanide solution to produce an insoluble blue
compound, ferric ferrocyanide (Prussian blue).
Ferrocyanide solution
1% aqueous potassium ferrocyanide 20 ml
2% aqueous hydrochloric acid 20 ml
Preferably freshly prepared just before use.
Method
1.Take a test and control section to water.
2.Treat sections with the freshly prepared acid ferrocyanide solution for 10–30 minutes
3.Wash well in distilled water.
4. Lightly stain the nuclei with 0.5% aqueous neutral red or 0.1% nuclear fast red.
5. Wash rapidly in distilled water.
6. Dehydrate, clear, and mount in synthetic resin.
Results
Ferric iron blue
Nuclei red
Perls’ Prussian blue reaction Cont…

•Ferric ion = blue

•Nuclei = red
•Background = pink
 Hematogenous Cont…

B. Hemoglobin
 Hemoglobin is a basic conjugated protein that is responsible for the
transportation of oxygen and carbon dioxide within the bloodstream.
Demonstration of hemoglobin
-Two types of demonstration method can be used to stain hemoglobin in tissue sections.
- Benzidine nitroprusside methods- not routinely used because of carcinogenicity of
benzidine
- Leuco patent blue V method

Leuco patent blue V method

Working solution
Stock solution 10 ml
Glacial acetic acid 2 ml
3% hydrogen peroxide 1 ml
-Prepare immediately before use.
 Hemoglobin Cont…
Method
1. Take test and control sections to distilled water.
2. Stain in patent blue solution for 5 minutes at room temperature.
3. Rinse in distilled water.
4. Lightly counterstain in 0.5% aqueous neutral red or 0.1% aqueous
nuclear fast red for 1 minute.
5. Rinse in distilled water.
6. Dehydrate, clear, and mount in synthetic resin.

Results
-Hemoglobin peroxidase (red blood cells and neutrophils) dark
blue
-Nuclei red
Hemoglobin Cont…
 Hematogenous Cont…
C. Bile pigments
 Bile is a fluid that is made and released by the liver and stored in the
gallbladder. Bile helps with digestion.
 It breaks down fats into fatty acids, which can be taken into the body by the
digestive tract.
 Bile contains, Mostly cholesterol.

Demonstration of bile pigments

Modified Fouchet’s technique
Solutions
Fouchet’s solution
25% aqueous trichloroacetic acid 36 ml
10% aqueous ferric chloride 4 ml
-Freshly prepared before use.
 Bile pigments Cont…
Method
1. Take test and control sections to distilled water.
2. Treat with the freshly prepared Fouchet’s solution for 10 minutes.
3. Wash well in running tap water for 1 minute.
4. Rinse in distilled water.
5. Counterstain with van Gieson solution for 2 minutes.
6. Dehydrate, clear, and mount in synthetic resin.
Results
• Bile pigments emerald to blue-green
• Muscle yellow
• Collagen red
Gmelin technique
 This technique is the only method that shows an identical result with
liver bile, gallbladder bile, and hematoidin. The method tends to be
messy, capricious, and gives impermanent results. Deparaffinized
sections of tissue containing bile pigments are treated with nitric acid,
and a changing color spectrum is produced.
 Bile pigments Cont…
• Method
1. Sections to distilled water and mount in distilled water.
2. Place mounted section under the microscope using an objective with
reasonable working distance.
3. Place 2–3 drops of concentrated nitric acid to one side of the coverglass and
draw under the coverglass by means of a piece of blotting paper on the
opposite side.
4. Remove excess solution and observe pigment for color changes.
Results
-Bile pigments will gradually produce the following spectrum of color change:
yellow-green-blue-purple-red.
D. Porphyrin pigments
 These substances normally occur in tissues in only small amounts.
 They are considered to be precursors of the heme portion of hemoglobin.
 The porphyrias are rare pathological conditions that are disorders of the biosynthesis
of porphyrins and heme.
 ENDOENOUS PIGMENTS Cont…
2. Non-hematogenous endogenous pigments
A. Melanins
Melanins are a group of pigments whose color varies from light brown to black.
The pigment is normally found in the skin, eye, substantia nigra of the brain, and
hair follicles.
A number of methods can be used for the identification of melanin and melanin-
producing cells.
The most reliable of these are:
1. Reducing methods such as the Masson-Fontana silver technique and Schmorl’s
ferricferricyanide reduction test.
2. Enzyme methods (e.g. DOPA reaction).
3. Solubility and bleaching characteristics.
4. Fluorescent methods.
5. Immunohistochemistry (melanin activation antigens).
 Melanins cont…
Masson-Fontana method
Method
1. Take test and control sections to distilled water.
2. Treat with the ammoniacal silver solution in a Coplin jar that has been covered
with aluminum foil, for 30–40 minutes at 56°C or overnight at room temperature.
3. Wash well in several changes of distilled water.
4. Treat sections with 5% aqueous sodium thiosulfate (also known as hypo) for 1
minute
5. Wash well in running tap water for 2–3 minutes.
6. Lightly counterstain in 0.5% aqueous neutral red or 0.1% aqueous nuclear fast red
for 5 minutes.
7. Rinse in distilled water.
8. Dehydrate, clear, and mount in a synthetic resin.
Results
Melanin, argentaffin, chromaffin and some lipofuscins black
Nuclei red
Melanins cont…
 Non-hematogenous endogenous pigments Cont…
B. Lipofuscins
• These yellow to red-brown pigments occur widely throughout the body and are
thought to be produced by an oxidation process of lipids and lipoproteins.
• The lipofuscins react with a variety of histochemical and tinctorial staining
methods, 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.
 Lipofuscins cont…
Long Ziehl-Neelsen method
Fixation
Any fixative.
Sections
Works well on all types of tissue section.
Method
1. Slides to distilled water.
2. Stain in a Coplin jar with filtered carbol fuchsin using a 60°C water bath for 3 hours, or overnight at
room temperature.
3. Wash well in running water.
4. Differentiate in 1% acid alcohol until the background staining is removed.
5. Wash well in running tap water.
6. Counterstain nuclei with 0.25% aqueous methylene blue in 1% aqueous acetic acid for 1 minute.
7. Dehydrate, clear, and mount in synthetic resin.
Results
Lipofuscin magenta
Ceroid magenta
Nuclei blue
Background pale magenta to pale blue
Lipofuscins cont…
 ARTIFACT PIGMENTS
• These are deposits of artifactually produced material caused by the
interactions between certain tissue components and some
chemical substances, such as the fixative formalin.
• Formalin and malaria pigments are sometimes classified as a
subdivision of endogenous pigments.
• This group of pigments comprises:
• formalin
• malaria
• schistosome
• mercury
• chromic oxide
• starch
 EXOGENOUS PIGMENT
 These substances gain access to the body accidentally through a
variety of methods (e.g. carbon anthracotic pigment, which is seen
in lung and lymph nodes).
 These pigments and minerals serve no physiological function.
 Entry is gained either by inhalation into the lungs or by
implantation (e.g. tattoos) into the skin.
 Most exogenous pigments are minerals, few of which are
pigmented.
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
Tattoo pigment
 EXOGENOUS PIGMENT cont…
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.
 Histologically, brown granules are deposited in collagen, basement
membranes, nerve sheath, blood vessel walls, and elastic fibers.
Carbon
 This exogenous substance is the most commonly seen mineral in
tissues and is easily recognized in stained tissue sections.
 Commonly found in the lung and adjacent lymph nodes of urban
dwellers, the main sources of this material are car exhausts and
smoke from domestic and industrial pollution.
 Tobacco smokers also inhale particulate carbon, and also give
passers-by a small sample.
Carbon cont…
 EXOGENOUS PIGMENT cont…
Silica
• silica in the form of silicates is associated with the majority of all
mined ores, because they are found in, or near, rocks that contain silica.
• Mine workers can inhale large quantities of silica, which can give rise to the
disease silicosis.
• This disease may present as a progressive pulmonary fibrotic condition
which gives rise to impaired lung capacity and in some cases extreme
disability
Asbestos
• Asbestos has been used for many years as a fireresistant and insulating
material.
• There are twogroups of asbestos that cause pulmonary disease in humans:
-serpentine (curly fibers) and
-amphibole (straight/chain-like fibers).
 Summery
 Demonstration of pigment in tissue is an aid to
diagnosis
 Pigments may be present itself in tissue section
with variety of morphology
 It is advisable to note a pigment’s morphology,
tissue site and relevant clinical data, to carry
out various special stains for confirmation
 References
• Bancroft’s Theory and Practical of Histological
Techniques 7th edition

• The Science of Laboratory Diagnosis second

edition
 GROUP MEMBERS
Name ID no
1.Fasika Adugna
2.Fitsum Israel
3.Fuad Junid
4.Getahun Ayele
5.Gezahegn Yabiru 0985/13
6.Halima Mulaw
7.Hayat
8.Leta Kebede
9.Maruf Mohammed
10.Mahlet Yigletu