Microtomy

Question 1

Rotary microtome

Answer 1

• can cut 2-3 um thin sections
• good for a wide variety of tissue types (hard, fatty, fragile etc)
• can ribbon and do serial sections
• can be fully automated
• improve section quality
• increase productivity
• less chance of musculoskeletal disorder dev or techs — elbow, shoulder, wrist

Question 2

Sliding microtome

Answer 2

• block is stationary and knife moves
• good for:
  
  • large blocks
  • hard sections
  • can do whole mounts (placing a whole organism or specimen on a slide for exam) (neuropathology, ophthalmic pathology) (RESEARCH)
• cannot cut as thin as rotary microtome
• cannot ribbon
• can do serial sections

Question 3

Knives

Answer 3

• EM used glass and Diamond because they cut VERY thin on plastics
• plastic techniques for bigger sections eg bone use Ralph knives (type of glass knife)
• has tool edge

Question 4

Disposable blades

Answer 4

• need sharp, defect free edge; dull/nicked knives cause artifacts
• big or low profile - type chosen depends on microtome blade holder being used
• advs. :
• reliable sharpness
• ease of use
• high and low profile available
• don’t have to sharpen

Question 5

CUTTING ANGLES (slide 7)

Answer 5

1. Bevel
   - very tip of knife
   - 27- 32 degrees
2. Wedge
   - main knife angle
   - 15 degrees
3. Clearance
   - angle between bevel and block face
   - 2–5 degrees
4. Rake
   - inverse of clearance angle
   - 90degrees minus clearance minus bevel angles

Question 6

Clearance angles issues

Answer 6

A. Tilt too slight
- missed or skipped sections
- alternating thick and thin (vibrating against block )
- wrinkled tissue
- tissue lifted on upstroke

B. Tilt too large (knife tip almost perpendicular to block); less support for knife
- chatter
-microchatter
- washboarding
- may not be able to get a ribbon

Question 7

Floatation bath

Answer 7

• should be able to control temp (kept at 10 degrees below melting point of paraffin)
• must keep bottom free of bubbles (clean with tissue wipes, coin, slide)
• for compression issues can add surfactant
  
  • alcohol
  • detergent
• most places can use tap water but some places have too much rust, sulfur or endemic microorganisms in the water; use distilled water
• must be kept clean
  
  • can grow mold and bacteria
• clean with soap and water after each use

Question 8

Drying oven

Answer 8

• goal is to remove water between section and slide
• If not done tissue will not stay adhered to slide; floats off during sectioning
• not to melt the paraffin
• if using horizontal staining need to let slides drain vertically before start heating
• temp should be around or a little above melting point of parafffin
• if too hot can cause distortion (>70deg)
• dark pyknotic nuclei ( misrepresentation of state of tissue since this usually marks apoptosis)
• nuclear bubbling from steam made by water pushing the nuclear proteins around
• cells completely devoid of nuclear detail
• usually 60 deg for 20-30 min
• can do 37 deg for 1 to 2 hrs could let it sit overnight for best results
• can do room temp overnight (aka air drying)
• can be done on newer platform automated H&E stainers

Question 9

Slide Types

Answer 9

• variety of sizes
• routine : 76* 25mm, 1-1.2 mm thick

Section adhesives - used when tissue may detach from slide
- exposure to strong alkaline solution during staining
- frozen sections
- special procedures eg IHC, IF or special stains
- sections that will be exposed to hot or cold temps (oven)
- CNS tissue
- tissue with increased blood and mucus
- hard and crunchy tissue (esp decalcification tissue)

Question 10

Adhesives

Answer 10

• gelatin (Knox or capsule), albumin (egg white) and starch : prone to microbial growth and increased background staining
• poly L-lysine: bought as a solution, diluted out 1:10 with water; slides are dipped in solution to coat the allowed to dry; only good for few days after being made
• 2-amiopropylthiethoxysilane (APES or Silane coated) : slides dipped in 2 % solution of APES in acetone, drained dipped in acetone, drained again, rinsed in distilled water and dried upright; good for cytology specimens that are bloody or have increased amount of protein
• pre-manufactured-charged slides
  
  • commonly used, have a permanent positive charge
  • utilizes a polymer that contains amino groups to bind and stick to silicone in glass
  • esp good for IHC antigen retrieval methods
  • will go bad, need to rotate stock and check expiration dates

Question 11

Sectioning artifacts/issues

Answer 11

most common issues:
- dull knife
- something loose
- wrong tilt

Question 12

Ribbon not forming

Answer 12

1. Wax too hard:
   - hard wax (high melting point paraffin; get lower melting point paraffin)
   - breath on block to warm up
   - don’t place on ice
   - cut at room temperature
2. Wrong clearance angle
   - knife clearance angle
   - too small or to large >15<
   - readjust

Question 13

Compression

Answer 13

1. Dull blade: move blade to a new area/ change blade
2. Soft wax: use harder paraffin/ice block
3. Dense tissue: ice block longer

Question 14

Ribbon comes off in a “C”

Answer 14

1. Top and/or bottom of block
   - not parallel to knife, trim with safety razor
2. Dull blade over part of block
   - compression of block in dull area
   - non compression in sharp area
3. Tissue is more dense in one area
   - less dense in other
   - more compression on dense side
   - ice the block

Question 15

Sections attach to block face on return stroke

Answer 15

1. Too small of a clearance angle; make larger
2. Tissue embedded at the top of block
3. Top of block “ragged”
4. Block vertical, not clear knife
5. Static electricity

Question 16

Sections roll up on knife edge

Answer 16

1. Dull knife (move or replace)
2. Sections too thick
   - make thinner section
   - use lower melting point wax =softer
3. Too large clearance angle
   - make smaller
4. Too sharp knife
   - brains
   - dull the knife (dull block or edge of pencil)

Question 17

Chippers/divots/moth eaten

Answer 17

Rough trim too aggressively
- knife pulls chunks of wax from block
-rough trim at smaller intervals
- do not use fist 2-4 sections on ribbon

Question 18

Thick and thin sections

Answer 18

See on on floatation bath where you have difference in size and shades of white in sections
1. Something loose
- tighten all levers and knobs
2. Too small clearance angle
- increase clearance angle
3. Wax too soft
- use higher melting point paraffin
- ice block

Question 19

Thick and thin sections

Answer 19

1. Tissue too dense for wax
   - overprocessed (cut down on time or temperature )
   - wax too soft (ice block; use higher melting point paraffin)
   - tissue naturally dense (uterus/ muscular structure)
2. “Light-weight” microtome
   - need a sturdier microtome or blade/knife
3. Hand rhythm
   - need to be uniform
   - not speed up, slow down
4. Faulty microtome
   - least likely
   - call repair person

Question 20

Venetian blind/washboarding

Answer 20

• thick and thin zones within each section
• visible with eye after staining

1.something loose
-not as loose as thick and think
- tighten all levers and knobs

2. Too much knife tilt
   - reduce clearance angle
3. Tissue is too dense for wax
   A. tissue is naturally dense: uterus, cervix, prostate
   - ice block
   - use higher melting point wax

B. Over processed
- reduce time or temperature of processing

C. Wax too soft
- ice block
- use higher meting point wax

Question 21

Microchatter

Answer 21

• thick and thin microscopically
• most often seen in:
  A. Epithelial layers of GI
  B. Fluid cysts
  C. Thyroid

Question 22

Microchatter troubleshooting

Answer 22

1. Something loose
   - not as likely
   - tighten everything
2. Tissue slightly hard
   - same actions as overprocessed
3. slow down
   - hand movement too fast; do even slow rotations
4. Tissue slightly over-processed
   - removed too much bound H2O
   - running biopsies on long cycle
   - correct:
   A. Soak block face in water for 5-60 secs
   B. Reduce processing time
   C: reduce temp on processor stations

Question 23

Tear/split/knife line

Answer 23

1. Nicks in knife edge
   - be carful removing sections from knife edge
   - previous/ current section/ bloc has something hard in it
   - line goes through entire section
2. Something hard in paraffin
   - particle of dust, dirt
   - remove from block
   - filter wax before use in embedding station
3. Something hard in tissue eg.:
   - calcium = use surface decal (good probs in breast and kidney cause it guides the docs to where the bad tissue is)
   - keratin = use surface NairTM
   - suture = metal or cloth
   - glass, sand, vegetative material
   - tissue component = keratin layer, muscle
   - cut thru last

• line starts on hard particle
• may eventually become entire line
• remove hard particle (probe or forceps)

Question 24

Tear/ Crack

Answer 24

1. Pulling on tissue hard
   - laying on water bath
   - removing from knife
2. Splitting along tissue lines (eg blood vessels, connective tissue splits from squamous tissue)
   - flotation bath too hot;
   - too long of time on water bath
3. Face plate holding blade
   - may be uneven
   - catches tissue sections

Question 25

dragging = raking on micro sections

Answer 25

- commonly seen in connective tissue layers (lymphatic tissue) - dull knife - to correct, move, replace blade

Question 26

Parched earth (seen in liver, placenta, blood vessels containing blood)

Answer 26

1. Random cracks in different directions - seen in: - overprocessed tissues - bloody tissue - lymphoid tissue - reduce time/temp of processors - cryogenic spray To correct: soak block - water - water + glycerin - water + dish detergent - water +wetting agent

Question 27

Folds/ wrinkles

Answer 27

1. Poor technique - anchor at one end - remove with forceps - pick up other sections 2. Curved tissue E.g. artery, appendix Solution: ended straight, leave on flotation bath a longer time 3. Too hot flotation bath - wrinkles/ folds melt in place - not enough time to remove wrinkles - solution = cool water temperature

Question 28

Waves

Answer 28

- 3D old - seen in curved tissue - action: increase temp of flotation bath; increase time on flotation bath =make sure to clean water bath as well because endemic bacteria can grow in it