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HTL Certification > Processing > Flashcards

Processing Flashcards

1
Q

What are the steps in tissue processing?

A
  1. Fixation
  2. Decalcification (if required)
  3. Dehydration
  4. Clearing
  5. Infiltration
  6. Solidification
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2
Q

What factors affect processing?

A
  1. Agitation - increased solution mvmnt+ increase reagent flow into specimen
  2. Heat - increase temp = increase rate of penetration and fluid exchange
  3. Vacuum - removes reagent in exchange for next reagent (removes trapped air and good for dense/fatty tissue)
  4. Viscosity of reagents - smaller molecules = less flow resistances into tissue = lower viscosity and reagents permeate quicker
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3
Q

Fixation

A
  • stabilizes and hardens tissue with minimal cellular distortion
    -inadequate cross linking/coagulation = macro molecular rearrangement during subsequent steps ~ smudgy nuclei, nuclear bubbling etc
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4
Q

Decalcification

A
  • removes mineralization of calcium in tissue (only performed on specimens with a large amount of calcium)
  • 20:1 ratio of volume : tissue
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5
Q

What are the factors that determine the decalcifier?

A
  • urgency
  • size
  • degree of mineralization - how hard is it?
  • scope of investigation - can we put it in molecular studies?
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6
Q

Types of decalcifying techniques

A
  1. Acid : strong acids (HCl, nitric), weak acids (formic), ion-exchange resin, electrolytic methods
  2. Chelation : EDTA, preserves DNA/RNA for subsequent molecular studies
  • acetic and picric acid will decal microcalcifications during fixation
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7
Q

Dehydration

A
  • pulls free water from tissues
  • most infiltrating media is immiscible with water
  • if tissue is not fully dehydrated = clearing agent can’t penetrate = infiltrating media cannot penetrate/solidify tissue properly = under-processed tissue
  • don’t want to overdehydrate by removing naturally bound h20 = micro chatter
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8
Q

What reagents are used in dehydration?

A

Alcohols, acetone and universal solvents

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9
Q

How do you select dehydration reagent?

A
  1. Agent selection: must be miscible with the reagent the tissue is in previously and following
  2. Tissue type: delicate tissue should start in lower levels of alcohol (30-50%) and normal in higher levels (70-80%)
  3. Fixation: non-aqueous fixative tissues should start in 100% (prevents water entering tissue thru osmosis/hypertonicity)
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10
Q

How do you store dehydrated tissue?

A

Can safely store in 70% alcohol without morphology changes
- anything lower, tissue will swell
-anything higher, tissue will shrink and become brittle

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11
Q

1: Properties of Ethanol

A
  • clear, colorless and flammable
  • not safe to consume as reagent due to addition of isopropanol and methanol
  • miscible with water and may contain 1-2%
  • used in gradations
  • can add eosin/phloxine to 100% to dye tissues pink for better visibility in embedding and microtomy
    - may cause autoflorescence
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12
Q
  1. Properties of Methanol
A
  • aka wood alcohol
  • evaporates quick, unpleasant odor, toxic
  • rarely used as a reagent
  • used in hematology and cytology as fixative - blood smears, cyto preps, Diff-quik staining

Poisonous thru skin; ingestion = blood stream = liver= formaldehyde + formic acid = blindness and death

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13
Q
  1. Properties of isopropanol
A
  • retains moisture ~ 1% water in it
  • doesn’t overharden tissue
  • good substitute for ethanol for paraffin infiltration (cannot be used for celloidin technique, not miscible with nitrocellulose)
  • cannot be used for some staining solutions = eosin
  • mild irritation and toxicity if ingested
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14
Q
  1. Properties of butanol
A
  • not common in lab
  • requires longer dehydration time = acts slower
  • has pronounced odor
  • good for animal and plant work
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15
Q

Alcohol substitutes for dehydration

A
  1. Ethylene glycol - cellosolve
    - not easily found
    - rapid dehydrant
    - not used in graded percentages
  2. Propylene glycol ether (doesn’t fix tissue)
    - pro-soft
    -not used in graded %
    - will not remove bound water = easier for microtomy
    - will not fix tissue and cannot be used for staining, not in processing
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16
Q
  1. Properties of Acetone
A
  1. Rapid dehydrant
    - but can cause excessive shrinking and hardening
    - easily removed by clearing agents
  2. Rapid evaporation ( watch volumes closely)
  3. Less expensive than alcohols
  4. Volatile
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17
Q

What are universal solvents?

A

They are reagents that dehydrate and clear (2 in 1)
- cannot be used with delicate tissue
Common reagents are: (all toxic)
A. Dioxane
B. Tertiary butanol
C. Tetrahydrofuran (THF)

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18
Q

Describe dioxane

A
  • faster than ethanol
  • less shrinkage than ethanol; leave in tissues for longer periods than ethanol
  • if water is left in tissue = more shrinkage
    -very toxic, pronounced odor, carcinogen
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19
Q

Describe tertiary butanol

A
  • pronounced odor
  • expensive
  • may solidify at room temp
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20
Q

Describe tetrahydrofuran

A
  • miscible with many solvents, water and paraffin
  • acts rapidly w/o shrinkage or hardening
    -least toxic of group but can cause dermatitis and conjunctivitis
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21
Q

What is a clearing agent?

A
  • called that because it made tissue look translucent
    -removes alcohol and sets tissue up for infiltration; must be done with proper dehydrant for adequate infiltration
  • clearing agents remove lipids
  • don’t leave tissue in for too long or else microchatter will occur
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22
Q

How do you pick reagents for clearing?

A
  1. Speed at which it removes dehydrate
  2. Ease of inliftration
  3. Flammability
  4. Toxicity
  5. Cost

Need to be disposed of by hauling away

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23
Q

Refractive index

A

Tissues with different refractive indices (RI) will have light bend and it will appear hits
Tissues with similar RIs will appear clear/translucent

24
Q

Name the clearing reagents:

A
  • aromatics hydrocarbons : benzene, xylene, toluene
  • chloroform
  • essential oils
  • limonene
  • aliphatic hydrocarbons
  • universal solvents (dual role of dehydration and clearing)
25
Q

Properties of xylene

A
  • mixed isomer, hydrocarbon
  • clears quickly to endpoint
  • time in reagent needs to be controlled, can easily harden tissue
  • toxic (neurotoxin that can cause a headache, dizziness, lack of coordination, mental confusion
    OSHA PEL = 100 ppm, STEL = 150 ppm
26
Q

Properties of toluene

A
  • Aromatic hydrocarbon, similar to xylene
  • clears to translucency and has safety risks
  • will NOT overharden tissue (tissue can stay overnight; recommended for *brains, muscle and tendon *
  • smaller molecule than xylene so evaporates quicker = more toxic fumes
    OSHA PEL= 50ppm
27
Q

Properties of Benzene

A
  • aromatic hydrocarbon, similar to xylene
  • clears to translucency, serious safety risks
  • carcinogen of bone marrow, SHA PEL = 10 ppm
  • tissue hardening is in between toluene and xylene
  • evaporates rapidly - do not need to change paraffin as frequently
28
Q

Properties of chloroform

A
  • chlorinated hydrocarbon
  • penetrates slow but far (needs more time than xylene)
  • causes less brittleness than aromatic hydrocarbons (good for uterus, muscle, tendon)
  • rapidly evaporates, use in a well ventilated area; toxic= toxic gas, phosgene and is a known carcinogen
  • different RI than tissue, will it create translucency
29
Q

Essential oils

A
  • very slow to clear - used for special research or long term storage
  • can go from 95% alcohol to essential oil ( must remove with xylene or toluene before using for microtomy)
  • very strong odor, use in well ventilated ares
    E.g:
    • cedarwood
    • clove
    • sandalwood
    • wintergreen
30
Q

Limonene

A
  • terpene is marketed as a xylene substitute
  • by product of citrus (generally regarded as safe; techs can become sensitized to citrusy smell)
  • safer than aromatic hydrocarbons
  • compared to xylene: slower to clear by 1.5 times, hardens tissues must change paraffin more frequently (guess its bigger molecule)
  • similar to xylene - cannot dump down the drain, must be hauled away
31
Q

Aliphatic hydrocarbons (alkanes)

A
  • xylene substitutes
  • proprietary chemicals - alkanes (short chain hydrocarbon (between paraffin and octane)
  • safety : non irritating, non sensitizing, low inhalation toxicity
  • compared to xylene:
    • take 2x as long
    • not tolerant of water (rotate alcohols more)
    • not miscible with all mounting media ( not recommended for automated coverslippers
  • disposal = waste hauler/ recycling
32
Q

5. Infiltration

A

Exchanging clearant fluid with fluid media that will be used for embedding and subsequently support upon solidification

33
Q

Types of infiltrating media (must know)

A
  • paraffin
  • water soluble waxes
    • polyethylene glycol (PEG)
  • celloidin
  • plastics — used for electron microscopy and hard tissue
    • glycol methacrylate
    • epoxy resins
34
Q

Paraffin

A
  • Most common
  • excellent for large output and diff types of tissue
  • infiltrates with aid of vacuum (increase infiltration speed)
  • commercially bought and has additives
35
Q

Paraffin additives

A

A. Beeswax - reduces crystal size and increases stickiness
B. Rubber - reduces brittleness, increases stickiness and ease of sectioning
C. Bayberry wax

Hardness increased by Ceres in, stearic acid, diethylene glycol diesterate, microcrystalline waves, plastic
- each paraffin will vary amount of additives to vary cutting properties (every company is different)

36
Q

Paraffin properties

A
  • high melting point = harder paraffin; better support for harder tissues and thinner sectors
    • more difficult to ribbon
  • lower melting point = softer paraffin; less support for harder tissues
    ~ easier to ribbon

Routine work done with paraffin is usually between 55 - 58 degrees C

  • too much time in paraffin - will cause shrinkage and hardening
  • paraffin should be kept 2-4 degrees C above melting point
    ~ in processor, if it goes above, tissue will harden
    ~ in embedder - cutting properties of paraffin will change
37
Q

Advantages of paraffin

A
  • cheap
  • easy to use
    -ribbons well
  • non toxic
  • thin sections ~ 2um (one cell layer)
  • easily stored
  • works well on automated processors
38
Q

Disadvantages of paraffin

A
  • requires heat
    • removes water
      • shrinkage, hardening, distortion (microchatter)
    • inactivates enzymes and some antigens that will be required further down the line in IHC f.e
    • temp must be controlled
  • can’t be used for electron microscopy because the sections aren’t small enough
  • messy
39
Q

Polyethelene glycol (PEG) (not used routinely)

A
  • aka carbowax
  • water soluble wax
  • can go from fixative into graded percentages of alcohol
    • have to heat it to melt
  • can go form any percentage alcohol into PEG (from 95% don’t have to worry about shrinkage)
  • can cut moderately think sections (3-4um)
  • doesn’t ribbon as well as paraffin
40
Q

How to float PEG in waterbath

A

PEG is water soluble so add:

  1. Potassium dichromate and gelatin to waterbath
  2. Diethylene glycol, formaldehyde and carbowax to waterbath
  3. Place directly on slide and then into oven
41
Q

How to store PEG blocks

A
  • hygroscopic blocks
    ~ store in plastic bag with small amounts of desiccant (to absorb water molecules)
42
Q

Celloidin

A
  • nitrocellulose compounds used from embedding
    ~ most common = parlodion
  • process:
    • from 95% EtOH tissue is placed in a 1:1 of ether and ethanol
  • infilatrated though graded% of celloidin dissolved in the 1:1 ether and ethanol
  • starts at 2% and embedded in 12%
  • celloidin hardens thru evaporation of ether ethanol solution to the consistency of gelatinous mix, or gum drop then hardened using **chloroform* (good for brain tissue)
  • sections held in 80% ETOH until stain and then mounted on glass slides
43
Q

Advantages

A
  • no heat required (shrinkage and hardening are minimal
  • good for brain sections
44
Q

Disadvantages

A
  • takes a long time (weeks to months)
  • cannot cut thin sections or serial sections
  • requires explosive reagents
    • anhydrous ether
    • nitrocellulose
45
Q

Resins and Plastics

A
  • hardest of the embedding media
  • support hard tissues and cut very thin
  • types :
    • glycol methacrylates
      • can support cutting un-decalcified whole bone or ceramic implants (pathos study this to see surrounding infection etc)
    • epoxy resins
    • cut very thin sections for electron microscopy (600-800 angstroms)
46
Q

SOLIDIFICATION

A

Process of transitioning from a liquid to a solid state

  • crystallization (paraffin and carbowax)
  • evaporation (celloidin)
  • polymerization (resins and plastics)
47
Q

A. Crystallization

A

Paraffin and carbowax

  • media is solid at room temp - heated to turn to a liquid
  • embedding media is called rapidly to create a solid structure with a small crystal size
    • small enough for light microscopy
    • too large for electron microscopy
    • if cooling is not done fast enough - will result in large crystal size
48
Q

b. Evaporation

A

Celloidin

  • cannot use heat - would prematurely harden media
  • takes a long time — days to weeks to months depending on size of block (ether ethanol solution has to evaporate )
49
Q

C. Polymerization

A

Resins and plastics
- except celloidin

  • liquids are combined together to react and create solid matrix
  • electro microscopy — epoxy resins
    • heat used to speed up solidification
  • histology -glycol methacrylates
    • exothermic rxn -heat is generated during polymerization
    • usually done on ice to prevent tissue from overheating
50
Q

Microwave processing

A
  • more commonly used for small tissue biopsies
  • can be used for large specimens
  • utilizes heat to speed up processing

Fixation —> isopropanol —> paraffin

51
Q

automated processors and schedules

A
  • can add heat, agitation and vacuum to decrease processing times
  • Custom programs can be created for different tissues
    • small, short runs for biopsies
    • standard runs for larger tissue
    • extended runs for fatty tissue - breast and brain
  • can use the delays to end process at specific times/days (will hold in fixative until time to start)
  • containment of fluids and fumes
    • some will even calculate management of reagents so you can replace
  • open system = carousel and rotates for replacements
  • closed system - tubing sucks agent from retort reagents
52
Q

Tissue processor QC

A
  • Reagent volumes should be maintained to cover sections in retort chamber
  • schedules created on an indiv. Lab basis
    • higher vol labs done on reg. basis
    • smaller vol labs done after certain # of runs (tissue vol.)
    • charts should be maintained to keep tract on how often the machine is run ad how often it is cleaned (hot water flushes)
    • solutions should be rotated not dumped; first solution dumped and replaced with new reagent; last 2 pushed forward
  • temp of processor, paraffin pots and embedding centers kept (can aft how paraffin ribbons form; can destroy additives if not monitored)
    • low vol labs should replace paraffin after a week if not used
  • buffering salt precipitate can be removed with cute acetic acid or thru preventative measures, eg how water flashes
53
Q

Tissue processing schedules

A

Need minimal time for xylene and paraffin for the following (to prevent over hardening)
- hard tissue
- muscle
- fibrous tissue
- scar tissue
- spleen
- bloody tissue
- small biopsies
- delicate tissue
- animal tissue

54
Q

Note difference in processing schedules of normal tissue vs biopsies

A

Biopsies:
- do not have to be fixed/fixed for too long
- less alcohol gradations
- less time spent in graded alcohol solutions
- less time spent in other solutions compared to other tissues;

Biopsies process for 2-3 hrs while normal tissues go 6-8 hrs

55
Q

Underprocessed tissue causes

A
  • wrong time in processing solutions
  • wrong schedule chosen — biopsy vs normal tissue
  • mix of routine and biopsy cassettes eg placenta in GI biopsies
  • xylene substitute using xylene processing times
    • xylene substitutes need 1.5 - 2 times longer
  • solutions place in wrong place in processor
  • switching of 95% and 100%
  • solutions not changed often enough
    • carry over from previous solutions; H2O presence can dilute graded alcohols — 100 to 95, 95 to 90 etc
    • paraffin contains too much xylene - prevents crystallization
56
Q

Correcting the prob- fixing underprocessed tissue

A
  • need to go backwards the reprocess
    What’s left in the tissue?
  • xylene - need a fresh change of paraffin
  • alcohol - go into paraffin and xylene
    -water - go into paraffin, xylene and absolute alcohol
  • several methods; where is it decided?
    • embedding, microtomy or have pathology decide
  • reprocessing will look better than original specimen but not as good as getting it right the first time

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