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Assignment
Topic: Special Stains
Submitted to: Dr. Maji Jose
Submitted by: Muhammed Ramshad.P
1 BDS
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Iron (hemosiderin)
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Hemosiderin (storage iron granules) may be present in areas of old hemorrhage or
be deposited in tissues with iron overload (hemosiderosis is the term used if the
iron does not interfere with organ function; hemochromatosis refers to a condition
of iron overload associated with organ failure).
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Perl's iron stain is the classic method for demonstrating iron in tissues. The section
is treated with dilute hydrochloric acid to release ferric ions from binding proteins.
These ions then react with potassium ferrocyanide to produce an insoluble blue
compound (the Prussian blue reaction). Mercurial fixatives seem to do a better job
of preserving iron in bone marrow than formalin.
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Hemosiderin, liver, iron stain.
• Calcium
• Only calcium that is bound to an anion (such as PO4 or CO3) can be
demonstrated. Calcium forms a blue-black lake with hematoxylin to give a
blue color on H&E stain, usually with sharp edges.
• VonKossa stain is a silver reduction method that demonstrates phosphates
and carbonates, but these are usually present along with calcium. This
stain is most useful when large amounts are present, as in bone.
• Alizarin red S forms an orange-red lake with calcium at a pH of 4.2. It
works best with small amounts of calcium (such as in Michaelis-Gutman
bodies). The alizarin method is also used on the Dupont ACA analyzer to
measure serum calcium photometrically.
• Azan stain can be used to differentiate osteoid from mineralized bone.
• Urates
• Uric acid crystals are seen in acid urine. In tissue, urates are present as
sodium urate. They are soluble in aqueous solutions and slightly soluble in
weak alcoholic solutions. Therefore, tissues must be fixed in 95% or
absolute alcohol to prevent leaching of urates.
• Methenamine silver stains urates black. Sodium urate crystals are also
birefringent on polarization. Using a red plate, the crystals show negative
birefringence (yellow color) when the crystal's long axis is aligned in the
direction of the slow wave. At 90 degrees to this, the crystals will be blue.
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• Uric acid crystals, polarized, with red plate.
• Copper
• The rare autosomal recessive disorder known as Wilson's disease
results from decreased serum ceruloplasmin, the blood protein that
transports serum copper. This leads to excessive copper
accumulation in brain, eye, and liver. Hepatic copper accumulation
results in fatty change, acute hepatitis, chronic hepatitis, and
eventual cirrhosis. Urinary copper excretion is increased.
• The rubeanic acid and rhodanine stains are utilized to detect the
cytoplasmic accumulation of copper in the liver.
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• Copper accumulation in liver, medium power microscopic.
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Exogenous pigments and minerals
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These come from industrial or environmental exposure by inhalation, ingestion, or contact. Sometimes exposure comes from work-related activities (miners). Sometimes they are
planned (tattoo).
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Carbon appears as anthracotic pigment in the lungs. It can be distinguished from melanin by doing a melanin bleach. Poorly fixed tissues may contain formalin-heme pigment, which is
black and finely granular, but this is widely scattered in the tissues without regard to cellular detail. Formalin-heme pigment is also birefringent on polarization.
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Asbestos is a special type of long-thin silica crystal, usually of the mineral group chrysotile. In tissue, these crystals are highly irritative and highly fibrogenic. The fibers become coated
with a protein-iron-calcium matrix, giving them a shish-kebab appearance. These are called "ferruginous bodies" because they are highlighted with an iron stain.
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Asbestos body, unstained.
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Asbestos bodies, iron stain.
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Silica is present in many minerals and building materials. Most forms are very inert and cannot be stained in tissue but can be demonstrated by white birefringence on polarization. It is
most often present in lung, but can make its way into lymph node.
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Silica crystals in silicosis of lung, polarized.
Street drugs for injection often are diluted with compounds containing minerals such as silica or talc. These crystals can be found throughout the body, but especially in lymphoreticular
tissues.
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Polarizable crystals in lung with intravenous drug use.
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Tattoo pigment is usually black and is inert and non-polarizable. Red tattoo pigment often contains cinnabar (which has mercury in it).
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Tattoo pigment in dermis of skin, H and E stain.
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In general, minerals are best demonstrated by microincineration techniques or by scanning electron microscopy with energy dispersive analysis (SEM-EDA).
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Another use for SEM-EDA in forensic pathology is for analysis of gunshot residue. The primer residue has a characteristic pattern because of the elemental composition which contains
antimony, barium, and lead.
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SEM-EDA pattern of gunshot residue.
• Fat stains
• The oil red O (ORO) stain can identify neutral lipids and
fatty acids in smears and tissues. Fresh smears or cryostat
sections of tissue are necessary because fixatives
containing alcohols, or routine tissue processing with
clearing, will remove lipids. The ORO is a rapid and simple
stain. It can be useful in identifying fat emboli in lung tissue
or clot sections of peripheral blood.
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• Oil red O stain of fat emboli in lung.