Pigments, Minerals &Cytoplasmic Granules Flashcards
Pigments
Visible substances in tissue found either naturally or as a result of a disease process
Pigments may be:
Artifacts
Exogenous
Endogenous
Artifact pigments
Produced by a chemical reaction with a tissue component
Most common are those caused by fixation
Acid hematin
Mercury pigment
Chrome pigment
mercury pigment
Caused by mercury containing fixatives
Must be removed with iodine
Iodine is removed with hypo (sodium thiosulfate)
Mercury containing fixatives include B5,Zenker & Helly
Mercury pigment cannot be prevented
Chromate Pigment
Easily prevented, wash tissues after fixation
Most authors feel it cannot be removed but some say acid alcohol will remove it
Examples of fixatives: Zenker, Helly, Orth
Formalin Pigment
One of three acid hematins ( other 2) :
Malarial pigment
Hydrochloric acid pigment
Characteristics: Birefringent Microcrystalline, dark brown No iron reaction prevented by buffering in neutral pH Dissolved with alcoholic picric acid or Alcoholic alkalis *
Exogenous Pigments
**Most common is carbon, a black substance resistant to bleaching and extraction with sulfuric acid – anthracotic pigment
Asbestos- birefringent fibers which become coated with iron containing protein & known as asbestos bodies. Asbestos bodies stain with Prussian Blue
- look like dumbells
Exogenous substances
Tattoo pigments are found in skin & occasionally in proximal lymph nodes
Minerals are not pigmented but some may enter the body & lead to disease
Examples: silica, lead, beryllium, aluminum, silver (fillings, piercing) See your notes
Endogenous Pigments
Hematogenous ( comes from blood) :
- Hemoglobin
- Hemosiderin
- Bile
Non-hematogenous ( doesn’t come from blood) :
- Lipofuscin ( wear & tear ) - looks the same as bile so need to differentiate bc liposfusion is harmless but bile is bad
- Melanin( skin, hair, eye color)
All of these pigments appear as yellow to brown
Hemoglobin
endogenous hematogenous
Hemoglobin, normally confined to RBCs may be found in areas of hemorrhage or hemolysis
Stains with Leuco Patent Blue
Not performed regularly
hemosiderin
endogenous hematogenous
Iron stores in the body consist of hemosiderin
Increased in disease:
Hemachromatosis, Hemolytic anemia
Stained with Prussian blue
bile pigment
endogenous hematogenous
Bile Pigment (bilirubin) may accumulate in liver when there is an obstruction
Staining involves converting bilirubin to biliverdin using trichloracetic acid in fouchet’s reagent
The green of biliverdin exhibits the bile pigments
bilirubin–(trichloracetic acid in fouchets reagent)–>bilirverdin(green)
lipofuscin
endogenous- non hematogenous- lipidic
Lipofuscin- “wear & tear” pigment ( harmless)
Found in heart, liver, & neurons of older persons
Stains with oil-red-o & sudan black ( bc of these are lipid stains) & PAS
Negative with bile stain
Ceroid
endogenous - non- hematogenous lipidic
Ceroid is rarely seen in humans
Stains with oil-red-0, sudan black, PAS but also is acid-fast
Melanin
endogenous non- hematogenous non- lipidic
Melanin is derived from tyrosine
A brown-black pigment
Normal in skin, hair, retina, iris & parts of CNS
Normal melanin may obscure the view of other tissue components
Melanin is removed by:
Peroxide (bleaching)
Strong alkali
Melanin is not removed by:
Weak acids or bases
Organic solvents
- looks like carbon but carbon isnt removed with bleaching & is removed w/sulfuric acid
( dont need to know) Melanin synthesis involves the formation of dopa (3,4 dihydroxyphenylalanine)- as an intermediary step. This can be used to identify melanocytes histochemically
melanin - turnbull reaction
The solubility of melanin can be used for identification
Melanin is strongly basophilic and reduces silver nitrate to metallic silver (argentaffin)
Gives a positive Schmorl reaction bc its argentaffin
Complexes with *ferrous iron which may be demonstrated by the *Turnbull reaction
- ferric is most common form of iron in the body not ferrous
Endogenous Minerals- Urates
Urates-deposited around joints in persons with uric acid metabolism disorders. * gout
Accumulate to form “tophi”
- Painful gout
Urates are water & lithium carbonate soluble
- Lithium carbonate extraction as control
Alcohol fixation or frozen section required For identification using either method
Birefringent
- Polarization may be used to ID
- Also used to examine body fluid ( uric crystals)
Methenamine silver stain
- With & without lithium carbonate treatment may be used
endogenous minerals- Calcium
Abnormal deposits are found in necrotic areas of tissue (salts of phosphates & carbonates) calcium is often with salt not usually stained alone i
Stains purple-blue with H & E
von Kossa silver method
Alizarin red
Cytoplasmic Granules
Group of body cells which exhibit common processes related to
hormone synthesis*
All these cells have a high uptake of amine precursors and the ability to decarboxylate
(APUD cells )
These cells are demonstrated by argyrophil / argentaffin reactions
- Adrenal chromaffin cells
- Pancreatic endocrine cells
- Gastrointestinal enterochromaffin cells
- “C” cells of the thyroid
- Neuroendocrine pituitary cells
fixation & preserving cytoplasmic granules
Chromaffin is best preserved by chromate containing fixatives (Orth)
Argentaffin GI granules are destroyed by alcohol fixation
Paneth cell granules are destroyed by acetic acid
APUD staining
While these cells may be stained using the metalllic impregnation methods( massson trichrome ) in this section they are generally stained today using immunohistochemistry
Prussian Blue Stain
A true histochemical reaction
Prussian blue is not a dye but a reactant colored product
Ferric iron is normally found in tissues
Large deposits in Hemachromatosis or Hemosiderosis
Reagents are potassium ferrocyanide & HCL
Ferric iron forms an insoluble bright blue pigment
Do not confuse with ferrous ions (Turnbull reaction)
Prussian Blue Stain steps
Hydrate
Handle with non-metal forceps
Mix 2% Pottassium Ferrocyanide & 2% Hydrochloric acid half & half,
heat 20 min at 60°C.
Wash with distilled
Counterstain (Nuclear fast red, Neutral red Eosin)
Rinse , DCM
Prussian Blue stain results
Results
Nuclei and hemofuchsin…….Bright red
Hemosiderin (iron)…………….Blue
Background ……………………..Pink
Precautions for Iron Stains
Be sure glassware is chemically clean
- Beware of iron Hematoxylin or ferric chloride
Iron may be dissolved by acid fixatives or decal
- False negative
Control with limited iron content is desirable to avoid possible background staining
Turnbull Blue
For ferrous iron not to be confused with ferric iron
Ferrous iron in tissues is extremely rare
This reaction is however used for the Schmorl technique
The reaction is different, we use ferricyanide not ferrocyanide to produce ferrous ferricyanide
Prussian vs Turnbull Blue
PRUSSIAN
Detects the usual iron storage compound
Detects ferric iron (hemosiderin) Fe3+
Uses potassium ferrocyanide
Used also for colloidal iron
TURNBULL
Detects a rare iron storage compound
Detects ferrous iron
Fe2+
Uses potassium ferricyanide
Used also for schmorl technique
Schmorl technique used on
Used to indicate the presence of reducing substances in tissue
Melanin
Argentaffin granules
Formalin pigment
Schmorl Technique for reducing substances
Employs the Turnbull reaction
Staining solution contains ferric iron
Used to detect reducing substances in tissue
Reducing substance reacts with ferric iron to form ferrous iron
Ferrous iron reacts with ferricyanide to produce ferrous ferricyanide (Turnbull blue)
May be used with Mayer’s mucicarmine for GI (mucin is stained)
May be used without the mucicarmine & mentanil yellow steps & counterstained with nuclear fast red instead
Same precautions as Prussian blue ( non metal forcepts)
May be used for melanin (argentaffin)
Generally replaced with IHC
Fontanna-Masson steps
Hydrate section & appropriate control Immerse in silver nitrate, 1 hour 56°C Rinse in DI Tone Fix, counterstain, DCM
Fontanna-Masson
An argentaffin technique for melanin or certain cell granules
Any reducing substance will give a positive reaction
Also replaced generally by IHC
Overstaining causes background staining & loss of contrast *
When staining for melanin, use two slides & bleach one. (potassium permanganate or peroxide)
This makes the method more specific
Argyrophil Stains
Grimelius & Churukian are two silver methods for detection of granules in neurosecretory tumours
Both use hydroquinone as a reducer & are counterstained with nuclear fast red (kernechtrot)
IHC is a more accurate means of ID
GMS for Urates
Must be alcohol fixed bc water & lithium carbonate soluble
Same principle as fungal stain
Stained a longer time
Frozen section and polarizer could be used instead
Bile Stain
Ferric chloride in trichloracetic acid (Fouchet reagent) oxidizes bilirubin to biliverdin which is a green color.
van Gieson’s is used as a counterstain
Differentiates lipofuscin & bile (bilirubin)
Bile or bilirubin ……..green
(emerald to olive)
Backround ……………yellow
Calcium Staining
von Kossa – an interesting metallic impregnation technique that uses light as a reducer
Alizarin Red S – a dye which forms a chelation complex with calcium
H&E stains calcium purple - blue
Von Kossa
actually a carbonate and phosphate staining method
calcium is found in tissues attached to these substances
Indirect staining method
von Kossa stain steps & results
Hydrate
Place sections in silver and expose to light
Rinse in DI
Fix in hypo
Counterstain in nuclear fast red
Wash, DCM
RESULTS…….calcium- black
background- redAlizarin Red S
Using a pH of 4.1 – 4.3 for this staining method is important
At this pH the stain is said to be specific for calcium (by some)
This pH may cause the localization of the calcium to be diffuse
Copper Staining
Seldom required
WILSON’S DISEASE*** is quite rare, deposits usually in liver
Methods used include:
RHODAnine (more sensitive, less specific)
RUBEANic acid (Uzman’s) less sensitive, more specific
Shikata Orcein (probably not as sensitive)
