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Histology > Tissue processing > Flashcards

Tissue processing Flashcards

1
Q

what are the steps in tissue processing

A

dehydration

clearing

infiltration

embedding

  • fixation should be complete before processing begins. if not add fixation time to your processing schedule
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2
Q

dehydration

  • main use
  • how we prepare it
  • also used for
A

removes water from tissue
-hydrophilic reagents attract water from tissue

gradual increase in concentration used
- known as graded alcohols

dehydrates are also used for tissue sections as part of staining process

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3
Q

what is the most commonly used for dehydration

A

Ethanol ( ethyl alcohol )

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4
Q

what happens if you don’t use graded alcohol ( dehydrate all in one step )

A

tissue distorted

especially in delicate tissue

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5
Q

closed processors should start with processor with no greater than

A

approx 60% to avoid precipitated buffered formalin in the lines or even worse tissues

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6
Q

when can dehydration be accomplished in one step

A

microwave dehydration can be done in one step bc molecules are in motion and therefore rapid diffusion throughout specimen is possible

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7
Q

when may dehydrates be used beside for processing

A

dehydrates are also used fro tissue section sa part of staining process

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8
Q

Ethyl alcohol advantages

A 
non-toxic(relatively) 
miscible 
little shrinkage with graded alcohols 
may be used for eyes or embryos if graded 
fast acting 
reliable
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9
Q

ethyl alcohol disadvantages

A 
expensive 
long exposure can cause shrinkage and hardening 
record keeping ( every mL)
theft prevention 
extracts some dyes from tissue
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10
Q

safety - alcohols

A
  • flammable( usage, storage &disposal )

ethyl is intoxicating
methyl & isopropanol are poison

violet reaction with oxidizing agents including silver nitrate

> 24% needs waste management
24 or less discard does sink

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11
Q

isopropanol advantages

A

penetrates as well as ethanol

good substitute for ethanol

less shrinkage & hardening than ethanol

less expensive

no government restrictions

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12
Q

isopropanol disadvantages

A

not suitable for stain preparations such as eosin (eosin is insoluble in it)

cannot be used for celloidin

nitrocellulose is insoluble in it

mildly irritating

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13
Q

acetone advantages

A

rapid dehydration
less expensive
doesn’t remove dyes from stained sections

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14
Q

acetone disadvantages

A

requires 20X that of tissue

flammable- low flashpoint

excessive shrinkage

best graded with with xylene before paraffin

volatile

melts pastic coverslips ( not recommended for automatic cover slippers)

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15
Q

methyl alcohol advantages and disadvantages

A

works well

may cause blindness or death

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16
Q

denatured alcohol aka methylated spirit

A

ethanol with 1% methanol added

works well

not subject to alcohol taxations

security measures not needed

no record keeping

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17
Q

which alcohol is the most important

A

the last alcohol is the most important
- water content should be <2%

verify alcohol is anhydrous by adding alcohol to xylene
- water turns white in xylene (cloudiness)

specific gravity may be used to determine water content

desiccant may be added (calcium or copper sulfate ) as an indicator of water
- BUT not suitable for pump action processors

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18
Q

added to dehydrates

A

softeners

  • phenol
  • mollifex( alcohol/glycerin mix)
  • dish detergent in DI water

dye

  • eosin ( NOT with isopropanol) or alcian blue
  • may tiny tissue more visible and easier to embed
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19
Q

universal solvents

A

same reagent achieves both dehydration and clearing

advantage is speed

disadvantage is extreme toxicity, cost, and unpleasant aroma

ex. dioxane, tertiary butanol or tetrahydrofuran

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20
Q

what two processing reagents must a universal solvent be miscible with ?

A

fixative ( usually aqueous)

paraffin

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21
Q

clearing meaning and must be miscible with

A

removing alcohol from tissue and replacing it with clearing agent
- dealcoholization

must be miscible: dehydrating alcohols, infiltrating media such as paraffin, mounting media such as permount (slides)

many change the refractive index in tissue

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22
Q

incomplete dehydration or clearing

A

prevents paraffin from infiltrating and= mushy blocks

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23
Q

too long in clearing agent

A

makes tissue too brittle

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24
Q

criteria for choosing clearing agents

A

speedy removal of dehydrant

ease of removal by molten wax

minimal tissue damage

flammability

toxicity

cost

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25
Q

Xylene & substitution protocol

A

one of the best clearing agents & is used widely

changing to a new agent would require splitting specimens , processing with both clearing agents and comparing the results with a large number of antibodies

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26
Q

xylene advantages

A

clears quickly

makes tissue transparent, endpoint obvious

will not dissolve cellodin

does not affect aniline dyes

turns cloudy in presence of water

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27
Q

xylene disadvantages

A

toxic

prolonged treatment over hardened tissue

hazardous waste

  • removed by licensed waste disposal $$
  • or may be distilled (recycled)
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28
Q

xylene safety

A

xylene is a neurotoxin

requires ventilation

can cause CNS damage

flammable

gloves

automatic cover slipping reduces exposure

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29
Q

toluene advantages

A

doesn’t harden like xylene ( great for CNS aka nerve & brain )

best aromatic hydrocarbon

clears quickly

makes tissue transparent

clear endpoint

more tolerant to water than xylene

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30
Q

toluene disadvantages

A

TOXICITY

addictive

dermatitis, headaches & dizziness

long term exposure harmful to organs

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31
Q

benzene advantages

A

fast acting

makes tissue transparent

clear endpoint

hardens less than xylene

quick evaporation from paraffin

less shrinkage than xylene & toluene

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32
Q

Benzene disadvantages

A

proven carcinogen !

very volatile

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33
Q

chloroform advantages

A

less hardening than xylene

not flammable

better for uterus, muscles & tendon ( hard tissue )

penetrates well

works for celloidin

34
Q

chloroform disadvantages

A

absorbs water from air

slow

volatile

***may damage vacuums pump membranes

disposal $$

doesn’t change refractive index

violent reaction with acetone

35
Q

acetone advantages

A

boils off at 58 degrees, less wax contamination and changing

dehydrates and clears

36
Q

acetone disadvantages

A

volatile( great ventilation needed)

flammable

***shrinkage ( best graded with xylene)

volume needs 20x that of tissue

interfere with some automatic cover slipping

37
Q

essential oils advantages

A

clears quickly

less hardening than other s

little evaporation

38
Q

essential oils disadvantages

A

expensive

must be removed with xylene

smells may be nauseating

oil residue makes cutting difficult

39
Q

which essential oil if the best known

A

cedar wood oil

40
Q

xylene substitutes ( first wave Limonene)

A

“goo gone”

hardens less than xylene

citrus odor

more changing of paraffin$$$

greasy

waste disposal

may cause problems with mounting slides

41
Q

xylene substitute ( second wave - aliphatic hydrocarbons- alkanes) advantages

A

low toxicity

combustible

gentle on tissue

42
Q

xylene substitutes( second wave ) disadvantages

A

intolerant to wattle ( humidity )

not compatible with all mounting media

three station on processor ( xylene need just 2)

deparaffinizing section needs 3 sections instead of 2

43
Q

infiltration

A

supporting media holds cells & intracellular stricture in proper relationship

paraffin ( best choice )

water soluble wax

celloidin

plastics

epoxy

44
Q

paraffin is most popular

A

cheap

easily handled

relatively easy section production

shorter processing time

45
Q

paraffin additives

A

beeswax - stickiness

rubber- reduces brittleness

other waxes - smooth texture, smaller crystals

plastic - increases hardness & support

46
Q

commercially supplied paraffin

A

increased melting temp = hard wax
- thinner sections ( ribboning is difficult )

decreased temp= softer wax

  • better ribboning
  • harder to get thin section & less tissue support
  • most labs use lower melting point ( 55-58) in an effort to preserve antigens for immunochemistry

temps must be monitored & adjusted when necessary

47
Q

paraffin on the tissue processor

A

tissue should be in paraffin for a short time to avoid shrinkage & hardening ( can cause more damage than overheating the embedder, oven or floatation bath )

wax should be kept at 2-4 degrees above melting point

change frequently to avoid clearing agent contamination

  • rotation of 4 baths
  • odor is a sign of contamination
48
Q

paraffin under pressure

A

vacuum speeds up process

removes residual air

*may damage tiny biopsies

pressure shouldn’t exceed 500mm Hg

49
Q

tissue processors

A
50
Q

tissue processors most popular

A

closed/fluid transfer

  • pumps each solution in and out
  • allows temp & pressure motion for each station
51
Q

microwave processors

A

becoming less popular

  • complete fixation before processing
  • reagents manually moved *
  • isopropanol used as “intermediary”(can dehydrate & clear )
  • wax is non-polar, & invisible to microwave radiation
  • allows temp & pressure option for each station
52
Q

tissue processing by hand

A

extremely urgent specimens

possible prion CJD infection

special circumstances

53
Q

new automated instruments may achieve full processing in less than one hour

A

has proprietary solutions, on board microwave, &thinner sections

sakura expresss

54
Q

processing protocol

A

fluid transfer -set allows embedding first thing in the morning

microwave -possibly at intervals during the day ( 2-3) runs

continuous throughput instruments - new batch every 40 mins

55
Q

what is the weakness of having all cassettes in fluid waiting to be embedded

A

tissues are all processed the same regardless if size or type

tiny biopsies may require shorter time in reagent

56
Q

operators can schedule processors

A

time, pressure & heat in each station

vacuum is usually applied to at least one station

57
Q

which processors can be used safely at 37 degrees

A

all except paraffin

58
Q

processing station and reagents

A
  1. 10% neutral buffered formalin ( stations 1-10, 35 degrees)
  2. 70% alcohol
  3. 80% alcohol
  4. 95% alcohol
  5. 100% alcohol
  6. 100% alcohol
  7. 100 % alcohol
  8. clearite 3
  9. clearite 3
  10. clearite 3
  11. paraffin ( 60 degrees)
  12. paraffin (60 degrees)
  13. paraffin (60 degrees)
  14. paraffin (60 degrees)
59
Q

when do you changed reagents

A

depends on

  • age of regents
  • # of blocks run
  • any obvious contamination

change in rotation not a full change

60
Q

why does alcohol on tissue processor require frequent changing ?

A

alcohol absorbs water from the tissues 7 humidity from the air

61
Q

sources of contamination ( carryover)

A

alcohol

  • water from tissues in aqueous vocative
  • water from air

clearing agent

  • alcohol
  • water if the last alcohol contains more than 2% *

paraffin

  • clearing agent
  • alcohol if clearing agent was not changed *
62
Q

other infiltration media

A

carbowax - water soluble in wax

celliodin - nitrocellulose compound

plastics - glycol methacrylate GMA

epoxy resin

63
Q

carbowax

advantages

A

fats can be demonstrated *

some enzymes can be demonstrated

no dehydration or clearing

64
Q

carbowax disadvantages

A

cannot be floated on water ; instead use ***

  • potassium dichromate & gelatin in water
  • diethylene glycol, gelatin, formaldehyde, carbowax

must be store in desiccated plastic bags **

will nit infiltrate if tissue is extremely fatty

65
Q

celloidin advantage

A

no heat used in processing **
- allows very delicate tissues to be processed without distortion

  • favoured for brain research **
66
Q

celloidin disadvantages

A

process ,may take weeks to months***
- gradual increases in concentration 2-14%

blocks must be stored in 80% alcohol ***

will tolerate no water

67
Q

plastics advantages

A

embedding of calcified bone ***
- cut with a glass knife

great cellular detail
-kidney, bone marrow & lymph h node

68
Q

plastics disadvantages

A

difficult microtomy

staining difficult

dangerous chemicals
- use under hood

69
Q

epoxy resin

A

used for electron microscopy ***
- diamond knife 60-90nm thick sections

avoid akin contact

70
Q

agar & gelatin

A

used for friable tissue

block may be then embedded in paraffin ( double embedding )

71
Q

30% sucrose

A

serves as a cryoprotectant

formalin fix, infiltrate with sucrose solution

dry slides well

tissue will sink when infiltrated

72
Q

OTC

A

water based solutions used to prepare tissue for frozen sectioning

leaves no discolouration of slide or tissue

allows cutting of small bone fragments
- this can be an advantage for diagnosis of possible bone tumor( decal& other infiltrates may damage making diagnosis more difficult )

73
Q

processor precipitation

A

too high alcohol concentration following NBF

zinc formaalin must have pH below 7.0

74
Q

over dehydration

A

evidence - microchatter in tissue **

process biopsy & large specimens separately

decrease dehydration time

75
Q

poor processing

A

evidence - poor to absent nuclear staining

water in tissue evident when placed in clearing agent

76
Q

poor processing solutions

A

change reagents more frequently

change fume hood control as recommended

may be condensation in processor

may be mechanical problems
use heat for paraffin only

77
Q

improper processing consequences

A

makes microtomy difficult

decreases the quality of the sides produced

78
Q

failure to follow SOP ( not defendable )

A

forgotten to load a solution

loaded solutions in wrong sequence

solutions not changed often enough

programmed incorrectly

79
Q

when errors occur

A

acknowledge
attempt remediation
file incident report
how could this be prevented in the future

80
Q

open processors

A

seldom used

have unique problems

81
Q

sponge artifact

A

sponges or lens paper may be used to secure tiny specimens in cassette throughout processing

sponges should be presoaked in fixative

use biopsy paper or lens paper

82
Q

specimen thickness

A

4mm or less in cassette

garbage in garbage out - no processing method works if sections are too thick for solution to penetrate

Histology (15 decks)

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