fixation, pigments, artifacts Flashcards
what are the functions of fixation
- prevent autolysis
- stabilize tissue morph
- enhance staining
how do fixatives work
altering tissue proteins by denaturation and forming crosslinks
what is denaturation
altering the shape of a protein (secondardy and tertiary structures)
how does heat denature
molecules vibrate rapidly = weaken chemical bonds
how does alcohol denature
disrupts hydrogen bonds and forms a bond between the alcohol and amino acid chains = tissue hardening
how do acids denature
hydronium ions react with amino and carboxy group and arginine, histidine, and lysine = break saltlinkages
how do heavy metals denature
affinity for sulfur in disulfide bonds (mercury) = secondary structures altered
what is crosslinking and how does it work
chemical reaction with amino acids by forming methylene bridges cross links = harden tissue and reduce shrinkage
what is the most common cross linking fixative
aldehydes like formaldehyde and gluteraldehyde
how do aldehydes react with tissue protein
First covalently bind to amino groups lysine, cysteine, serine, and threonine to prevent autolysis
then they bind to one another to form methylene bridges (slow process)
examples and fuction of oxidizing agent fixatives
osmium tetroxide and potassium dichromate
osmium reacts with lipids for EM
dichromate ion links to carboxyl
what are coagulant fixatives and how to they work
distort cytoplasmic content while stabilizing proteins
work fast compared to non coag
tertiary structure affected
what are non- coagulant fixatives and how to they work
preserve cytoplasmic content by turning into gel and stabilize by forming cross links
gel stops penetration of reagents = work slower
primary structure affected
additive vs non additive
additive = chemically binding proteins
non additive = no chemicals binding = dehydrating agents
examples of additive non caoag
formaldehyde, gluteraldehyde, osmium tetroxide, potassium dichromate
aldehydes and oxidizing agents**
examples of additive coag
mercuric chloride, chromic acid, picric acid, zinc salt
examples of non additive coag
ethanol, methanol, acetone
4 factors that affect quality of fixation
temperature
size
time
volume of fixative
what temperature should fixation be
<45C for light microscopy but routinely done at RT
<37 for EM
what size should tissue be for dixation
<3-4 mm thick
<1mm for gluteraldehyde
how long should tissue be fixed
minimum 8 hours using NBF
what volume of fixative to use
15-20X the tissue volume
how is formaldehyde prepared in histology
37-40% solution in water aka 100% formalin
used as 10% NBF that contains 4% formaldehyde
what is paraformaldehyde and how to prevent it
white precipitate from concentrated solution
prevented by adding methanol
how does formaldehyde work
cross linking with amino groups, arginine, histidine, and lysine
how does formaldehyde affect tissues
less shrinkage but hardens tissue when prolonged
fuction and use of gluteraldehyde
dialdehyde = crosslink while penetrating **tissue <1mm thin
used for EM
function and use of acetic acid
used to lyse RBC
cannot fix cytoplasmic contents therefore it is combines with other reagents
how does acetic acid affect tissues
penetrates tissue fast with no hardening
2 reasons why acetic acid is used in compound fixatives
preserves nucleoproteins and precipitates DNA
swells tissue
function and use of picric acid
both a fixative and stain
hydrolyze DNA/RNA
how does picric acid affect tissues
causes SEVERE shrinkage
decalcify small biopsies
storage of picric acid
stored wet as it can be explosive when dry
combined with metals = explosive so never pour down the drain
how is picric acid neutralized before tissue processing and what happens when it is not neutralized
use 70% ethanol or water
staining characteristics lost over time
Alcohol fixation affects on tissues
harden and shrinks
ethanol use for tissue
preserve glycogen and urate crystals
Use of osmium tetroxide
EM for lipids
What does bouins fluid contain
formaldehyde, acetic acid, picric acid
use for bouins fluid
trichrome staining
lysing RBC and decal
how is swelling from the picric acid countered in bouins fluid
using acetic acid
what does B plus contain
mercuric chloride, sodium acetate, formalin, water
uses for B plus
best for nuclear detail and rbc in IHC
instead of murcury in B plus, what is substitudted
zinc chloride
what is Clarkes fluide made of
3 parts ethanol, 1 part glacial acetic acid thats made fresh
uses for clarkes fluid
molecular pathology for nucleic acids and lipids
what is modified formalin made of
alcohol and formalin
uses for modified formalin
fixes and dehydrates
benefits from using modified formalin
prevents shrinkage
really good with fatty tissues like breast
why may zinc sulfate be added to modified formalin
preserve tissue antigenicity during storage
improve nuclear detail
what are endogenous pigments and give example
naturally occuring in tissue like melanin and lipofuscin
what is formalin pigment
birefringent dark brown in blood rich tissues
how does formalin pigment forms
hemoglobin reacts with formaldehgyde in acidic conditions
T/F formalin will create false positive in silver stains
T - formalin is an argentaffin (reduces silver)
what is murcury pigment
birefringent brown crystals that is non preventable
how is mercury pigment removed after fixation
iodine followed by hypo
examples of exogenous pigments
carbon - smoker lung
tattoo ink - dark and granular in skin
