w11 Flashcards
histology
study of microscopic structure of tissue
what is used as a fixative after biopsy or autopsy
10%v/v neutral buffered formalin to prevent degradation of tissue
when is tissue given accession number
once it arrives in history accessioning department
Histology accessioning
check patient info then accession it into LIS-generate barcoded labels for container, slides, and documents and a printer will put accession number onto cassette
s in accession number for surgical and a for autopsy, 2 digit for year then a case number.
4 steps in preparing tissue for microscope
- gross dissection
- tissue processing
- embedding
- sectioning
what is done during gross dissection?
pathologist or pathology assistant does it
-whole specimen examined and described for size, colour, texture and anomalies.
then representative sections are dissected and blasted into tissue cassette- remainder stored for 2-4 weeks
what goes on in tissue processing
cassettes loaded onto automated tissue processor which flood tissues in a series of solutions to fix, dehydrate, clear, and fill with wax
- fixation-formalin
- dehydration-alcohol
- clearing-xylene
- infiltration-paraffin
what occurs during the fixation process of tissue processing
preserves micro anatomy of tissue by preventing autolysis and bacterial decomp.- allows tissue to withstand treatment
done with 10% formalin (formaldehyde)
dehydration of tissue processing
removal of water, done with graded alcohol 70% then 80% then 95% then 100%
what is done during clearing process of tissue processing
-replaces alcohol with xylene- this is necessary because alcohol and paraffin are not miscible
infiltration of tissue processing
done with molten paraffin wax 60 degrees celsius
-replaces clearing agent, and completely permeates tissue and hardens
how VIP tissue processor works
-draw fluid by vacuum to tissue compartment then it is pumped back into storage container takes 1-2 hours or up to 16 hours
chemicals used in tissue processor
dangerous -hazardous fumes that are flammable, consult SDS and wear PPE - work in well ventilated room free of ignition -r95 can be worn.
what happens if volume of chemicals in tissue processor not sufficient
-poor or incomplete processes (dehydrating,clearing, infiltrating)
what to do if water removal incomplete (inadequate dehydration)? - tissue block soft and mushy tissue block
(carry over of water from incomplete dehydration the xylene will become murky and white)
to fix: reverse the processing steps to remove wax from tissue then repeat dehydration
melt was from tissue, then use xylene, then use alcohol, t
what is miscible in xylene
alcohol and paraffin
-it can remove both
what happens if alcohol is not completely removed and what to do
- it will prevent paraffin from penetrating tissues adequately and it will be soft and mushy and difficult to section
- reverse process to remove wax from tissue block and repeat, dehydration, clearing and infiltration with fresh reagents
tissue processor maintanence
temp checks on processing fluids- 40-45 degrees celsius. molten paraffin stored at 60
-purge cycle is after infiltrating tissue it removes paraffin for next batch (pumps xylene in and out of chamber multiple times)
embedding
done by MLT wax-infiltrated tissue placed in small metal mold- more paraffin used the end product is a block of tissue which is sectioned into 3-5 micrometers
sectioning
microtome slices tissue block 3-5 micrometers thick, they are floated out on warm water to remove wrinkles. then picked up on glass slide, placed in oven at 60 degrees to melt paraffin and fix slide
staining
reverse of tissue processing since distilled water is used so they are hydrated or brought to water
1.removal of paraffin wax with xylene
2. removal of xylene with alcohol most to least 100-70
3.water to remove alcohol
H&E hematoxylin and eosin used
hematoxylin- nuclei blue eosin- cytoplasm red
special stains
-demonstrate specific types of tissue, structures or chemical components within tissue
tips for good stain results
- check reagent expiry
- never allow slides to dry out
- keep reagents covered
- filter stains
- follow schedule for changing reagent
- monitor water quality
- microscopically check staining
- stain solutions accurately prepared
dehydration, clearing and mounting (occurs after staining)
- dehydration with graded alcohols to gradually remove water
- clearing agent is miscible with alcohol and with resinous mounting media like xylene
- mounting- applying cover slip this will protect stained tissue for storage and microscopic analysis
