Microtomy problems and how to solve them

Question 1

Crooked/uneven ribbons causes

Answer 1

-Horizontal edges (top and bottom) of the block are not parallel.
* Bottom edge of the block is not parallel to the cutting edge of the blade.
* Blade imperfections.
* Insufficient lateral movement of the blade when changing to the unused surface → the blade is “half dull-half sharp”.
* Uneven chilling of block.
* Variable hardness of the paraffin.

Question 2

Crooked/uneven ribbons CA

Answer 2

• Calibrate microtome to ensure the bottom edge of the block is parallel to the cutting edge of the blade.
• Check all surfaces of the blade; go to a new area of the blade; change blade.
• Lateral movement of the blade holder should be as wide or wider than the tissue block.
• Ensure all block faces are laid flat on ice and evenly chilled.
• Only one type of paraffin should be placed in the embedding reservoir and tissue processor.

Question 3

Block face unevenly sectioned causes and CA

Answer 3

Causes:
* Microtome was not calibrated - the specimen
clamp is not parallel to the blade holder.
* A major part of one side of the block has
been cut away because the entire block face is
not parallel to the blade.
* Unlevelled tissue block.

Corrective actions
* Calibrate your microtome to ensure the specimen clamp is parallel to the blade holder.
* Ensure all locking levers are engaged and the orientation screws are locked.
* Re-embed blocks that are unleveled
* Ensure cassette it sitting flat on the mold during embedding

Question 4

Holes in the section AKA – “Moth-eaten effect” causes and CA

Answer 4

Causes:
* Excessive dehydration (improper tissue processing).
* Aggressive coarse trimming.

Corrective actions
* Check the processing program – ensure adequate number of alcohol stations.
* Avoid aggressive coarse trimming. Gauge the distance of the block face to the blade surface.
* Smooth/polish the surface of the tissue by cranking the drive wheel (only) up to 10X. Ensure that the micrometer is set at 4µm when you perform this action.

Biopsies→ Be very conservative when coarse trimming these tissue blocks.

Question 5

Causes and CA of Failure of ribbon to form

Answer 5

Causes
* Dull blade.
* Warm block.
* Paraffin too sticky or too hard.
* Too much oil on the blade surface.
* Improper setting of clearance angle.
* Room temperature is too high or too low.

CA

• Chill block on ice for at least 10-15 minutes.
• Change the surface or change the blade altogether.
• Re-embed the tissue. If necessary, using the proper wax for the tissue you are sectioning.
• Wipe off oil from the surface of the blade using a thick wad of gauze.
• Ensure correct setting of the clearance angle.
• Call facilities to adjust the room temperature.

Note: If ribbon is unobtainable after checking and correcting the problems, pick up single section - except the first section.

Question 6

Section lifting from the blade as the
block is raised Causes and CA

Answer 6

Causes:
* Dull blade.
* Warm room.
* Paraffin too sticky.
* Clearance angle was too small.

CA
Change the surface or change the blade altogether.
* Call facilities to adjust the room temperature.
* Re-embed the tissue. If necessary, using the proper wax for the tissue you are sectioning.
* Ensure correct setting of the clearance angle.

Note: If ribbon is unobtainable after checking and correcting the problems, pick up single section - except the first section.

Question 7

MACROSCOPIC chatter AKA – “Washboarding or Undulations” Causes and CA

Answer 7

Causes
* Tissues that are dense, hard (E.g., uterus, cervix, bone) or over-fixed.
* Some parts of the microtome are loose.
* Clearance angle was too large.
* Inadequate block support.

CA
* Place hard tissue blocks in a solution to soften the tissues.
* Some parts of the microtome are loose –
* Ensure that the locking levers are engaged.
* The blade must be securely clamped and correctly loaded on the ledge.
* The tissue block must be correctly loaded and securely clamped on the block holder.
* Ensure correct setting of clearance angle.
* Ensure specimen holder is not over-extended (retract back).
* Ensure that the paraffin is filled to the top of the cassette to provide support for tissue when clamped in the block holder.

Question 8

MICRCOSCOPIC chatter AKA – “Venetian blind effect”
causes and CA

Answer 8

Causes
* Over-dehydration of the tissue.
* Lack of moisture in the tissue.
* Dull blade.
* Clearance angle was too large.
* Cutting too rapidly

CA
* Check the tissue-processing program. Ensure adequate
number of alcohol stations.
* Lack of moisture in the tissue – allow coarse-trimmed
blocks to soak in ice water for a longer period of time
before fine trimming.
* Change the surface or replace with new blade.
* Ensure correct setting of clearance angle.
* Cutting too rapidly – crank your drive wheel at ONE
revolution per second.

Question 9

Skipped or varied thickness AKA – “Thick and thin and Banding Causes and CA

Answer 9

• Clearance angle was too small.
• Bottom, instead of top, blade facet makes
  contact with the block.
• Loose knife or loose block.
• Loose or worn microtome parts

CA
* Ensure correct setting of the clearance angle so the top of the blade facet makes contact with the block.
* Loose knife or loose block – ensure knife is held firmly in
place and block is sitting securely in the block holder.
* Loose or worn microtome parts – your lab must ensure that preventative maintenance is done on a regular basis. Replace worn out or broken parts.

Question 10

Lengthwise scratches or splits AKA – “Scores” CAUSES AND CA

Answer 10

CAUSES
* Defect on the blade edge.
* Hard particle in the tissue.
* Paraffin buildup on the blade edge

CA
* Change the surface or change the blade altogether.
* Avoid any metal objects that may damage the blade edge.
* Paraffin collected in front or back of the blade edge – remove gummed up wax in between blade changes.
* Hard particle in the block – check for suture, staple, calcium or crystals.
* Pathological calcium must not be removed by surface decalcification.
* Crystals are identified before removing.

Question 11

Compressed or wrinkled ribbons
CAUSES AND CA

Answer 11

CAUSES
* Dull blade.
* Blade was gummed with paraffin.
* Paraffin sticking of the backside of the blade holder.
* Clearance angle too small or too large.
* Cutting too rapidly.
* Warm block.

CA
* Replace cutting surface – move to a new area of blade or change blade.
* Blade was gummed up with paraffin – ensure the blade was correctly loaded.
* Wipe off excess oil on blade to avoid wax sticking to it.
* Brush off wax sticking to the blade surface
* Paraffin sticking to the backside of the blade holder – remove gummed up wax using gauze (in between changing blades).
* Ensure correct clearance angle setting.
* Cutting too rapidly – crank drive wheel at one revolution per second.
* Call facilities to adjust room temperature.
* Place block back on ice and make sure it’s chilled before cutting.

Question 12

Sections flying and sticking to nearby objects or other parts of the microtome CAUSES AND CA

Answer 12

CAUSES
Static electricity.

CA
* Install humidifier in the lab.
* Avoid placing lab coats in plastic bags.
* Spray water mist in the room.
* Introduce humidity in the immediate cutting area.

Question 13

Tissue crumbling CAUSES AND CA

Answer 13

CAUSES
* Over-dehydrated tissue.
* Hemorrhagic tissue.

CA
* Hemorrhagic tissues - allow block to sit in ice water for a
minimum of 30 minutes.
* Do not attempt to cut a long ribbon .
* Use a good blade.

Question 14

Mushy center that will not cut CAUSES AND CA

Answer 14

CAUSES
Underprocessed tissue

CA
* Reprocess the tissue block as per lab protocol.
* Document somewhere that the patient block was underprocessed and will be delayed due to tissue re-processing

Question 15

what does the temperature of the floation bath have to be
what happens if too high or too low

Answer 15

• Temperature setting @ 5°-10°C below the wax MP.
• Too low - will cause folding artifact.
• Too high - will cause parched earth artifact from spreading.

Clean out the air bubbles with end of brush and clean surface with tissue
-Add adhesives if needed
-Use distilled water TO tap is okay but DNA studies need DH2O

Question 16

Parched earth artifact AKA – “splits” CAUSES AND CA

Answer 16

Causes
* Tissue was not processed properly.
* Floatation bath temp – too high.

CA
* Tissue was not processed properly
* Check tissue-processing program.
* Ensure adequate number of alcohol stations.
* Floatation bath temp – set @ 5°-10°C below the wax MP

Question 17

Peel off and folding over AKA – “splits” CAUSES AND CA

Answer 17

Causes
* Water film under the tissue section.
* Incomplete drying of tissue in the oven.
* Did not use adhesive.

CA
* Water film under the tissue section – allow slides to air dry after cutting and place them in the oven right after.
* Incomplete drying of tissue in the oven – at least 60 minutes at 60º C. Follow SOP on drying Histological preparations.
* Allow the tissue on the slide to dry out completely in the
oven before staining.
* Did not use adhesive – tissue sections might not adhere well on the glass surface.

Question 18

Too much adhesive - CA

Answer 18

• Use albumin or gelatin sparingly when coating the slide with it.
• Since albumin is a protein it will bind to the stain being applied onto the tissue.
• This renders excessive background staining on the tissue
  section

Question 19

Bubble under the section- CA

Answer 19

• Remove the bubbles at the sides and bottom of the waterbath before floating out ribbons on it.
• Try to float out the ribbon with one end free so that you can drag it towards you. This will alleviate trapped air underneath the ribbon.
• Do not pick tissues with this artifact

Question 20

Debris in waterbath- CA

Answer 20

• Clean the surface of the waterbath frequently.
• Always use fresh water at the beginning of microtomy.
• Do not float multiple tissue ribbons from different blocks.

Question 21

Debris in waterbath- CA

Answer 21

• Do not dip your fingers in the waterbath when picking up sections.
• Tie your long hair. If you have dandruff, cover your head with an O.R. cap.

Question 22

Section not flattened completely- CA

Answer 22

• Avoid picking up sections that have wrinkles on them.
• Use alcohol in your waterbath to help flatten ribbons.
• Crank slowly, ensure blade is sharp and tissue block is very cold.
• Skin sections must be dried adequately to avoid lift off during staining.
• Avoid scooping motion when picking up tissue section with a slide.
• Water film will remain underneath the tissue causing it to lift off the slide – another artifact.
• Do not place the slide in the oven immediately after picking up a tissue. Allow it to air dry first.

Question 23

Overheat drying (too hot) CA

Answer 23

• Avoid exposure to high heat.
• 80°C heat: there is a significant amount of shrinkage even if the exposure is minimal.
• 37°C overnight: you will notice that the shrinkage between the epithelium and the underlying lamina propria is minimal to non-existent.
• Similar result with slides that are allowed to air dry completely before they are placed in the oven at 60°C for 1 hr.

Question 24

Overheat drying (too hot for too long) CA

Answer 24

• High heat and prolonged exposure to high heat will result in nuclear bubbling or nuclear meltdown. Avoid this mistake like a plague.
• Nuclear components and chromatin patterns must be
  observable after all the staining procedure is done.