350 - Topic 2, 3, 5 (Gross Dissection, Fixation, Decalcification) Flashcards
What are the functions of fixatives?
- Prevent autolysis: alter enzymes so they are no longer effective; also toxic to bacteria and fungi which averts putrefaction
- Stabilize tissue morphology: preserve relationship b/w cells and extracell substances; also helps to maintain non-protein elements, esp. carb and lipids
- Enhance staining: expose more reactive sites on a protein to increase dye binding = makes stain more intense; some create additional reactive groups or even act as mordants
NOTE: not al fixatives improve staining (ex: formalin reduces available dye binding sites for an ionic dyes like eosin)
All histological fixatives work by …
Altering tissue proteins
Two major mechanisms of fixation:
Deanaturation
Formation of cross-links
What is denaturation ?
Alteration of the shape of a protein
- always involves 2ry and 3ry protein structures = largely result of hydrogen bonding, hydrophobic interactions, disulfide bonds and salt linkages
- heat, alcohol, acids, heavy metals
Heat (denaturation)
- molecules vibrate rapidly = disrupts weaker chemical bonds
- will produce random protein structure compared to chemical fixatives = reproducibility issues?
alcohol (denaturation)
- disrupt hydrogen bonding extensively
- net hydrogen bonds between alcohol and amino acid side chains = stabilizes denatured protein structure and hardening tissue markedly
- expose hydrophobic domains, unfolding polypeptide
How does alcohol remove water from protein?
They are hydrophilic and hygroscopic = will attract and absorb water
Acids (denaturation)
Supply hydronium ions in solution which readily react with amino and carboxyl groups in proteins as well as basic AAs (arg, His, Lysine)
- this action = breaks salt linkages, forms new ones = changes shape of protein
Heavy metals
- mercury, lead, zinc
- behave similarly to acids, though they have a particular affinity for the sulfur found in disulfide bonds (esp. mercury)
- disrupting disulfide bonds affects the secondary structure of many proteins
- metal react w/ neg-charged side chains and ionized carboxyl groups = forming insoluble precipitates
What do cross-linking fixatives do?
They chemically react with amino acids
- they stabilize morphology by forming methylene bridges bw adjacent reactive areas
- methylene bridge cross-linking harden tissue markedly and tend to reduce shrinkage during tissue processing
The most common cross-linking fixative
Aledehydes
- formaldehyde and glutaraldehyde
Reaction of aldehydes with tissue proteins
- Aldehydes covalent bind to amino groups, as well as lysine, cysteine, serine, and threonine residues; reaction occurs rapidly and prevents autolysis by rendering enzymes ineffective
- Adjacent tissue-bound aldehydes link with one another by forming methylene bridges; this cross-linking phase occurs more slowly = days or weeks to complete
Dialdehyde
Glutaraldehyde
- it cross-links and stabilizes as it penetrates tissue
Fixation using oxidizing agents
- osmium tetroxide, potassium dichromate
- poorly understood but known to stabilize tissues by cross-linking reactive groups in close proximity
- dichromate ion links adjacent carbonyl groups of many proteins; osmium readily reacts with unsaturated carbons (lipids)
Additive
Chemically binding with the proteins or other tissue component
Non-additive
Disrupting tissue structure without becoming chemically incorporated; ONLY dehydrating agents are non-additive (ethanol, acetone, methanol)
These are all additive fixatives
Aldehydes
Acids
Metal salts
Oxidizers
Factors affecting fixation (4)
- temperature: warmer the solution ,faster it will act on tissue
- size/thickness/ tissue type: different fixatives will penetrate at different rates; formalin (no more than 3-4 mm thick); glutaraldehyde (no more than 1 mm)
- time: duration of fixation varies considerably; minimize delay (prevent autolysis and putrefaction)
- volume of fixative: the higher the ratio of fixative to specimen vol, the better ALWAYS; use 15-20 times the tissue volume when aliquoting fixatives
Temp acceptable for light vs electron microscopy
Light = up to 45C
Electron = do not exceed 37C
All tissues should be fixed for a minimum of how many hours wen using NBF?
8 hrs
24 hrs or ore generally preferred
24 hrs for breast
T or F. Formaldehyde readily polymerizes in aqueous solutions
T! Concnetrated solutions of formaldehyde may form paraformaldehyde
Paraformaldehyde
- concentrated formaldehyde
- white powder which precipitates out of solution
- prevention: add methanol (around 10%) to concentrated solutions
- dilute formaldehyde does not polymerize
Unlike most other fixatives, acetic acid does not …
Fix cytoplasmic proteins
So never used alone; instead, used in compound fixatives
Why is acetic acid used in compound fixatives?
- it preserves nucleoproteins and precipitates DNA (coagulant fixative in this regard)
- it swells tissue more than any other fixative
This will hydrolyze nuclei acids and must be avoided if DNA/RNA analysis is anticipated
Picric acid
Why is it important tat picric acid is completely neutralized prior to tissue processing?
If any picric acid remains in the tissue after processing, expected staining characteristics of tissue will be lost over time
- 70% ethanol better than water for this
This is notable for preserving glycogen and urate crystals (gout)
Ethanol
- glycogen often detectable in formalin-fixed tissue due to trappings f large molecules within cells, but urates quickly dissolve in aqueous fixatives
Excellent fixative for tissue that will be stained with a trichromatic method
Bouin’s
- picric a cts as a mordant for an ionic dyes = exceptionally vivid
B5
Mercuric chloride
Sodium acetate
Formalin
Water
Excellent fixative for hematopoietic and reticuloendothelial tissues as nuclear detail will be exceptional
B5 or B-plus (zinc)
Clarke’s fluid
3 parts absolute ethanol with 1 part acetic acid immediately before use
Molecular pathology fixative
Clarke’s
Maintains microanatomical structures better than most other fixatives
Clarke’s fluid
Modified formalin
65-70% alcohol used as diluent for concentrated formaldehyde = alcoholic formalin
- alcohol speeds up tissue fixation and begins dehydration and formalin fixation helps prevent shrinkage associate with alcohol
- popular with large amounts of adipose (breast, colon)
This can be added to 10% NBF in order to preserve tissue antigenicity, esp. with prolonged storage
Zinc sulfate or chloride
- effect probably due to a reduction in ‘methylene brige’ formation
- addition of zinc also improves nuclear detail when compared to unmodified 10% NBF
an undesirable change in tissue created by lab’s attempts to process it
artifact
endogenous pigment
naturally occurring in tissue
- not artifactual
- melanin (granular dark-brown) and lipofuscin (yellow-brown)
how is melanin often demonstrated?
Fontana Masson stain
- may be bleached using potassium permanganate or other oxidizing agents
acid formaldehyde hematin
formalin pigment
formalin pigment
AKA acid formaldehyde hematin
amorphous, birefringent dark-brown black pigment
- noted in blood-rich tissues
- forms when hemoglobin reacts with formaldehyde in acidic conditions
- using NBF will minimize the amount of formalin pigment in tissue sections
T or F. formalin pigment will reduce silver solutions
T
it is an argentaffin substance
formalin pigment can potentially produce a false pos when trying to demonstrate spirochetes, fungi, melanin, reticulin
how do we remove formalin pigment ?
saturated solution of picric acid and alcohol followed by a running water wash
also alcoholic ammonium hydroxide works
a crystalline, birefringent brown pigment that may be found in any tissue fixed in mercury-containing fixative
mercury pigment
T or F. the formation of mercury pigment is non-preventable
T! all tissues fixed in mercury is treated with an iodine solution to remove the pigment, followed by 5% aq sodium thiosulfate (hypo) to remove iodine staining
stain precipitates
- on top of tissue
- can confirm by focusing in and out of focus
- avoided by filtering stains
- will form if stain is allowed to dry on the slide
exogenous pigments
- not naturally occurring
- must be introduced to patient’s body
- NOT ARTIFACTS
- carbon, tattoos
T or F. neither carbon or tattoo ink is easily removed from tissues
T!
tattoo usually does not obscure pathology
why is zinc formalin considered better than formalin for most tissues?
- prevents cross-linking; good for preserving antigenicity for IHC
- better nuclear detail
vapour fixative
ALDEHYDES
can continue fixing even as a gas
- useful for disinfection
how to differentiate between formalin pigment vs melanin
formalin = birefringent; can dissolve by alcoholic picric acid
melanin = only within cells; can remove by any oxidizers
decalcification
process of removing calcium from tissues
- some inherently calcified (bone)
- others = calcification is a pathological process
- removal of calcium required prior to sectioning paraffin blocks; if not = will not section = tears, holes, major disruptions
what do most labs use to decalcify tissue?
acids
- calcium salts soluble at pH 4.5 or less, so almost any weak or strong acid may be used
- formic acid combined with formalin is used routinely, and HCl is used when more rapid decalcification is required
strong acid for decalcification
hydrochloric, nitric
- 5-10%
- fast <48 hrs
higher danger
weak acid for decalcification
formic
5-10%
slow (3-10 days)
lower danger
T or F. tissue must be fixed prior to decalcification
T! if not, decalcifying agent will destroy delicate tissue components and alter tissue basophilia
- using formic aicd/formalin solution will allow simultaneous fixation and decalcification = ADVANTAGE
bischloromethyl ether
extremely hazardous carcinogen
- wash thoroughly after formalin fixation bc residual formalin may react with HCl to produce this
decalcification methods
- simple acid
- ion-exchange resins
- EDTA
simple acid to decalcify tissue
- submerge tissue in an appropriate decalcifying solution
- tissue should be suspended to allow to work on all surfaces
- agitation to prevent solution surrounding tissue from becoming saturated with calcium ions
- solution is discarded after saturation and replaced with fresh fluid
ion exchange resins to decalcify tissue
- improvement over simple acids
- resin will exchange ammonium inos for calcium ions preventing solution from becoming saturated = quickening decalcification and reducing need for solution replacement
- typically formic acid decalcifiers
EDTA to decalcify tissue
- selectively binds metal ions from the tissue without affecting any other tissue c components
- EDTA (neutralized with sodium hydroxide) is popular esp when acids might destroy labile tissue elements we hope to demonstrate = enzymes, molecular targets
- disadvantage = TAT; may trake weeks
under-decalcified
will not section well
over-decalcified
will demonstrate poor staining and morphology
three methods to detecting endpoint
chemical
radiography
physical
CHEMICAL ENDPOINT DETECTION
aliquot of used decal solution neutralized with ammonium hydroxide - reacted with ammonium oxalatae
any calcium ions present => insoluble calcium oxalate (white precipitate)
if calcium detected = assumed that calcium persists in tissue and is placed in fresh decal solution
if no precipitate forms = decalcification is complete
fats and accurate method
radiography endpoint detection
calcium is radio-opaque = solid white on x-rays
- x-raying specimens = easily see endpoint of decal
cost of equipment may be prohibitive for labs that do not regularly perform this
physical endpoint detection
endpt determined by bending, probing, or attempting to cut sample
- method is fast and relatively easy to perform, it is unreliable and small deposits of calcium may be missed
- this may also create artifacts in tissue; NOT RECOMMENDED
what happens after decalcification?
tissue may be further trimmed/sectioned
carefully washed in running tap water
submitted for routine processing
neutralization of residual acid is an important step to ensure good processing
some labs use lithium carbonate to ensure neutralization
the usual rot cause for over/under-decal
poor endpt detection methods
when do we recognize under decalcified tissue
during microtomy
- during sectioning = obvious scores, tears will e noted
- resistance with handwheel may be felt if large amounts of calcium present
- to correct = melt block to remove tissue and ‘reverse process’ = expose to xylene, absolute alcohol, graduated alcohols and running water
- place tissue in decal solution until complete
an easy and efficient way to remove small calcifications
surface decalcification
- if only a section or two is desired
- once block has been faced, place cut-side down in small amount of strong acid for 15-30 mins
- wash well in running water, chill, attempt to section once again
- acid will only act on exposed surface of tissue so try to get one of the first couple sections
how to recognize overdecal tissue
marked reduction in nuclear staining
- nuclei will appear pale red and contrast poorly with cytoplasm
- loss of basophilia
- difficult to reverse
- some basophilia may be restored by placing affected slides ina 5% solution sodium bicarb overnight
next morning = wash with water and re-stain
