Processing Flashcards
incomplete dehydration
accounts for the vast majority of processing problems and results in mushy tissue
alcohols
the most common dehydrating reagents
clearing agents
must be miscible with both the dehydration agent and the infiltration medium
primary purpose is to remove the alcohol used to dehydrate the tissue
prolonged clearing
results in hard, brittle tissues
xylene
most commonly used clearing agent, turns cloudy in the presence of water
toluene
does not over harden tissue as much as xylene
overdehydration
seen as microchatter in H&E stained slides, commonly seen in biopsy specimens, can be corrected by processing biopsies separately from other tissues and decreasing the amount of time in the dehydrating solutions
poor processing
evidenced by poor nuclear staining, often caused by water remaining in the tissue when it is placed in the clearing agent which results in poor clearing and infiltration, can also be caused by too much heat during processing
melting point
high melting point results in hard blocks that section thinly but ribbon poorly, while low melting point ribbons easily but doesn’t section as thin. 55-58C is common melting point
diagonal embedding
makes sectioning much easier
forceps metastasis
when fragments of tissue are transferred between blocks if the forceps aren’t wiped down between specimens
paraffin crystal size
smaller is better, allows for better support of the tissue
mushy in the middle
tissue was cut too thick during grossing and needs to be reprocessed
dehydrant
alcohol for removal of water
clearing agent
xylene to remove the alcohol and make the tissue receptive to paraffin, high index of refraction renders tissue transparent
infiltrating medium
paraffin infiltrates the tissue and gives it support
open tissue processor
rarely used because of reagent evaporation
closed tissue processor
preferred method
Ethanol (ethyl alcohol)
clear, colorless, flammable
fast, best dehydrant
hydrophilic
70-95-100% steps help reduce tissue shrinkage
Methanol (methyl alcohol)
clear, colorless, flammable, poisonous
rarely used
except for fixation of blood smears
Isopropanol (Isopropyl alcohol)
flammable, toxic
excellent substitute for ethanol
eosin can’t stain with it
doesn’t harden or shrink tissue as much as ethanol
Acetone
volatile, flammable dehydrates rapidly, cheap excessive shrinkage absorbs water when exposed to air hard to maintain solution levels in open air processors
xylene
most commonly used clearing agent
can overharden tissues
intolerant of water left in the tissue
flammable hazardous neurotoxin, do not pour down the sink
toluene
flammable, more volatile than xylene
does not overharden tissue as much as xylene
benzene
very volatile and toxic, carcinogen
fast acting, doesn’t overharden as much as xylene but not used in histology
chloroform
volatile, carcinogen makes tissue less brittle than xylene clears tissue slowly must use in tight containers because it easily absorbs water from the air best for uterus muscle and tendon
cedarwood oil
volatile, strong odor
hardens and damages tissue the least out of any reagent
clears quickly
used for special projects
limonene
xylene substitute
harden tissues less than xylene but contaminates teh paraffin more often
can’t dispose down drain
why is paraffin routinely used
large number of blocks can be processed in a short amount of time
easy to get serial sections as well as routine and special stains
water present during dehydration
results in mushy tissue and incomplete clearing and infiltration
how fixative pH affects the processing unit
if zinc formalin goes above pH 7 it can cause formation of precipitates
precautions for handling tissue processing reagents
gloves, most of the reagents are dangerous or irritants
importance of preventative maintenance on tissue processors
keep solutions clean so that there isn’t carryover of water causing underprocessing, maintain temperatures to prevent overprocessing and hardening of tissue
why do we decalcify?
because calcium deposits damage the microtome blade and make sectioning very difficult
why do we carefully monitor decalcification?
because it is essential to make sure the specimen is neither overdecacified nor underdecalicied since either state results in undesirable staining outcomes; no nuclear staining or left over calcium that stains very darkly and can damage the blade
decalcification
occurs after fixation but before processing
pros and cons of acid decalcifiers
fast is both a pro and con, need to carefully monitor the process so the sample isn’t over or underdecalcified
function of acid decalcifiers
acidic solutions between pH 3 and 5, calcium salts dissolve and then ionize, migrating into the surrounding solution. This is the most common type of decalcification in histology
determining the endpoint of decalcification
mechanical: test flexibility or scrape, no one does this method
chemical: the decalcifying solution is checked for the presence of calcium
radiography: x-rays are used to show that decalcification is complete
why do we remove decalcifying agents prior to processing?
to prevent carryover of acids that would interfere with processing
how to preform localized decalcification
once the block has been faced the surface can be placed in decal solution, then rinsed and sectioned to break down any calcium deposits that remain
Max size
2cm square, 3-4mm thick
