350 - Topic 6 (Tissue Processing & Embedding)

Question 1

Four distinct stages in tissue processing:

Answer 1

• fixation: fully fixed before further processing
• dehydration: remove all unbound water and fixative from tissue
• clearing: removes dehydrating agent and acts as solvent for paraffin wax; also raises refractive index of tissue closer to that of glass = high power magnification
• infiltration: support medium; paraffin wax = allows cutting of thin sections

Question 2

T or F. Dehydrating solutions are hydrophobic

Answer 2

F! hydrophilic! allows them to draw out water form the tissue and into the solution

solutions frequent exchanged with increasingly concentrated solutions which will dilute any remaining water

Question 3

ethyl alc as dehydrating agent

Answer 3

• routinely used
• infinitely soluble with water
• fast-acting
• non-toxic
• may cause hardening and shrinkage

Question 4

isopropyl alc as dehydrating agent

Answer 4

• miscible in water but IMMISCIBLE in salt solutions = tissue must be washed following fixation
• isopropyl can be mixed w paraffin so clearing agent not used/required
• not toxic
• does not harden/shrink tissue

Question 5

methyl alcohol as dehydrating agent

Answer 5

• similar to ethanol (ethyl alc)
• more hazardous/toxic

Question 6

What happens if a large concentration gradient exists between water and alcohol during dehydration?

Answer 6

cells may be distorted or damaged by flow of solutions
tissue should be subjected to 50-70% alc immediately after fixation, followed by increasingly concentrated solutions = 80%, 95%, then absolute alc

Question 7

what do clearing agents have in common?

Answer 7

they are soluble in both dehydrants and paraffin

Question 8

once tissue is dehydrated, tissue is filled with alcohol which does not dissolve in wax, so what does clearing agent do?

Answer 8

acts as a link between these steps = dissolves any remaining alcohol

leaves tissue receptive to non-polar substances = molten wax

Question 9

T or F. Clearing agents have a high refractive index

Answer 9

T! 1.4-1.51

helps make tissue appear transparent (1.51 to 1.62)

Question 10

xylene

Answer 10

routinely used
- fast-acting
- miscible with most solvents as well as paraffin
- over-exposure will harden tissue
- flammable, moderately toxic

Question 11

toulene

Answer 11

• similar to xylene; unlikely to harden tissues
• more volatile than xylene (more vapours)

Question 12

chloroform

Answer 12

• used for CNS that may become brittle when cleared w xylene; esp. brain and eyes
• non-flammable
• highly toxic
• in presence of oxygen = phosgene = exceptionally hazardous; poisonous gas

Question 13

Xylene substitute

Answer 13

• short chain aliphatic hydrocarbons; like butane and petroleum jelly
• less toxic and work almost as well as xylene
• intolerant of water so final alcohol must be completely anhydrous

Question 14

universal solvents

Answer 14

chemicals capable of dehydrating AND clearing tissue
two most commonly used = tetrahydrofuran (THF) and dioxane
neither recommended due to safety concerns and tendency to over-harden tissue

Question 15

lower melting points =

Answer 15

softer wax

Question 16

higher melting points =

Answer 16

harder waxes

Question 17

wax additives and their purpose:

Answer 17

• plasticizers: used to make wax harder and facilitate ribboning
• beeswax: lowers the melting point and makes section ‘sticky’
• rubber: facilitates ribboning by increasing elasticity
• resins: makes wax harder/increase the melting point

Question 18

factors affecting tissue processing:

Answer 18

agitation
heat
vacuum/pressure
viscosity

Question 19

how does agitation affect tissue processing?

Answer 19

• ensures solutions surrounding tissue do not become locally saturated
• helps avoid ‘dead zones’ where solutions are not effectively exchanged during processing
• most use magnetic stir bars; some blow air bubbles through solutions; other repeated drain and fill the retort

Question 20

how does heat affect tissue processing?

Answer 20

• heat will speed up; use w caution
  = sustained temps above 40C may alter microscopic appearance of delicate tissue
• most recommend no heat at al during F,D,C steps for highest quality and only enough heat to keep paraffin wax molten

Question 21

how does vacuum/pressure affect tissue processing?

Answer 21

• pressure = force solutions into tissues
• vacuum = open porous structures
• vacuum effective aid during infiltration; lowering pressure in retort = clearing agent more easily replaced by hot wax

Question 22

how does viscosity affect tissue processing?

Answer 22

• measure of how thick liquid is
• water is standard for low-viscosity
• alcohol and xylene = low-viscosity
• wax = significantly higher
• lower viscosity solvents work more quickly than higher = so timing in each station varied accordingly

Question 23

how does an enclosed tissue processor work?

Answer 23

• tissue remains stationary in retort
• reagents pumped in and out according to a programmed processing schedule
• reduces tech exposure to reagents and allows use of vacuum/pressure to speed processing/infiltration

Question 24

microwave processors

Answer 24

• speeds up processing and improve TAT
• heat tissues from the inside = speeds up diffusion of solutions
• processes in minutes
  -formalin and xyelene free = safer
• EXPENSIVE

Question 25

xylene-free processing

Answer 25

xylene substitutes - safer - aliphatic hydrocarbons dont clear alcohols as quickly so duration of clearing steps increased (up to 50%) - less tolerant of water contamination than xylene; anhydrous ethanol must be at least 97% pure to be effectively cleared some labs = mixture of isopropanol and ethanol; no clearing agnet at all - fixation - ethanol dehydration (50%) - continued dehydration/clearing (80% ethanol/20% isopropanol) - isopropanol clearing (100% isopropanol) - high temp paraffin infiltration = first wax bath should be held at 85C to effectively eliminate isopropanol

Question 26

this is used to monitor alcohol quality

Answer 26

hydrometer

Question 27

clean or purge cycle

Answer 27

before another processing cycle can begin, paraffin must be removed from the retort and lines of instrument flush lines w xylene (to dissolve wax) and then alcohol to dissolve xylene alcohol remaining in lines is no issue; can mix w fixative at beginning of next run xylene and alcohol used for cleaning are not used during processing and have non-rotating or dedicated positions

Question 28

most common cause of inadequate processing

Answer 28

incomplete dehydration - microtomy = soft, mushy area at centre of tissue (wax failed to infiltrate) = hole on the finished slide ; staining = interfere with nuclear staining overprocessed = esp small biopsies; brittle, and easily fragments when sectioning; microchatter is a frequent microscopic finding

Question 29

embedding purpose

Answer 29

- support medium for microtomy; thin sections - provides elasticity to resist compression caused by microtomy

Question 30

hard wax

Answer 30

- higher MPs - great support for harder tissues - better suited for cutting thin (2-3 um) sections

Question 31

soft waxes

Answer 31

- lower MP - easier to ribbon = ideal for serial sectioning

Question 32

appropriate mold size

Answer 32

2-3 mm bigger than tissue on all sides to ensure good support

Question 33

the most critical step in the embedding process

Answer 33

orientation

Question 34

tissue with lumens

Answer 34

cut and embedded to show a cross-section of lumen vas deferens fallopian tube appendix arteries ducts

Question 35

T or F. cut side down is what will be cut first

Answer 35

T

Question 36

tissues with layers

Answer 36

should be embedded on edge = all layers demonstrated on each slide skin intestine gallbladder

Question 37

embedding tissue with soft layers

Answer 37

embed so that tissue is oriented in a way that softest layer will contact blade first = reducing compression

Question 38

embedding elongated tissue

Answer 38

best to offset tissue slightly (up to 30 degree angle from horizontal) - important for tougher/harder tissues like bone and uterus to prevent compression-type artifacts

Question 39

T or F. Paraffin blocks are stored indefintely

Answer 39

T! wet specimens (in formalin) discarded after 8 weeks

Question 40

T or F. Paraffin blocks are stored indefinitely

Answer 40

T! wet specimens (in formalin) discarded after 8 weeks blockss retained for at least 30 yrs

Question 41

T or F. Paraffin blocks are stored indefinitely

Answer 41

T! wet specimens (in formalin) discarded after 8 weeks blocks retained for at least 30 yrs

Question 42

these are used to section undecalcified bone, teeth, or to cut thin sections (<2 um) for light microscopy

Answer 42

acrylic resins such as methyl methacrylate (MMA) or glycol methacrylate (GMA) methacrylates more convenient as the formulation can be varied to make a harder or softer block

Question 43

T or F. acrylic resins not useful for electron microscopy

Answer 43

T! they are unstable in the electron beam

Question 44

epoxy resins

Answer 44

used for electron microscopy also ideal for cutting semi-thin (0.5-1.0 um) ad ultra thin (~800 nm) sections

Question 45

3 rules to achieve high quality processing results

Answer 45

- must b completely fixed prior to dehydration - tissues must be thin <5mm to allow adequate penetration - processing schedules must be tailored to sizes and types of tissue processed

Question 46

high volume labs = reagent schedule

Answer 46

processor always full reagents changed on a set schedule

Question 47

lower volume labs = reagent schedule

Answer 47

reagents should be changed according to how much tissue has been processed

Question 48

potential causes of incomplete dehydration

Answer 48

- condensation (heat used during processing) - absolute alcohol is not absolute - wrong processing schedule - processor fault (mechanical failure)

Question 49

potential causes of over-processing

Answer 49

- excessive dehydration - processing biopsy samples using overnight/routine schedule - extended contact w paraffin wax

Question 50

crunchy tissue

Answer 50

over-processing

Question 51

variation in staining, periphery of specimen lighter than centre

Answer 51

zonal fixation formaldehyde binds to end terminal AAs as does eosin formalin fixation reduces eosin binding sites BUT if incomplete fixation = alcohol finishes fixation so more eosin binding sites = darker eosin in centre

Question 52

xylene on tissue processor is xanthochromic and turbid

Answer 52

water contamination turbid is the big thing! clear and yellow = ok! replace absolute alcohol as well