Microtomy

Question 1

What is the principle behind microtomy?

Answer 1

This process utilises a rotary microtome with a disposable microtome blade. The manual/ automated rotation of the laterally mounted wheel causes the advance mechanism to move the block holder towards the rigidly held stainless-steel blade at a pre-set section thickness. The paraffin wax block moves up and down through the blade in a vertical plane with the production of flat sections on the downward stroke

Once sections are cut, they are place in a spirit bath for manipulation of the tissue ribbon, and then they are floated on a warm water bath to help remove wrinkles. Then they are picked up on a glass microscopic slide.

The glass slides are then placed in a warm oven to help the section adhere to the slide

Question 2

What must be noted on each slide

Answer 2

All slides cut from paraffin blocks must be identified with the individual who cut the block and the microtome used to cut the block

Question 3

What tissue tend to cause problems at microtomy

Answer 3

hard or calcified material typically calcified prostatic chippings, microcalcifications in breast tissue, colloid, calcified heart valves, uterus, some skins and bone specimens that have not been adequately decalcified before processing.

Question 4

What might occur when trying to cut hard tissue

Answer 4

crunchiness, thick and thin sections, ‘Venetian blind’ effect, scoring or other artefacts may occur.

Question 5

What might be required for really hard to cut tissue and how is this done

Answer 5

RDC rapid decalcifier may be required to facilitate the cutting

The trimmed block is placed into RDC for ‘surface decalcification’ for a very short period of time.

Question 6

What tissue should never undergo surface decalcification

Answer 6

Trucut breast biopsies
Breast resection
Any tissue requiring IHC

Question 7

What are the seven steps to microtomy

Answer 7

Molds
Trimming
Cooling
Cutting
Floating
Collecting and drying
ID

Question 8

What is involved in the mold step

Answer 8

○ Wax blocks are cooled on a cold plate
The wax blocks must be removed from their molds before rough cutting

Question 9

What is involved in the trimming step

Answer 9

The cutting of the wax block up until a representative full cross-sectional area of tissue is obtained

Question 10

What is done in the cooling step

Answer 10

○ Place wax block back on cold plate
○ The wax will warm up very quickly when trimming
Block will need to cool down again before taking sections -> this will make microtomy a lot easier -> ribbons will be far easier to handle

Question 11

What is done in the cutting step

Answer 11

○ Use a separate area of the blade than was used for trimming (as trimming damages the blade)
○ Look at colour of casette to determine how many levels are needed:
▪ White blocks need one
▪ Orange need three + spares
○ Keep fingers away from blade at all times
○ Clean debris from blade using a small paintbrush -> brush away from the blade
○ Always use the blade guard when not cutting tissue
○ Always lock (chuck holder?) in place when not in use
Never trim the paraffin block so that there is no tissue remaining in the block unless specifically told to do so by the pathologist

Question 12

What is done in the floating step

Answer 12

○ Float ribbon onto spirit waterbath
▪ Alcohol reduces surface tension which allows ribbon to flatten out with ease particularly when transferred to heated water bath
▪ The warm water bath will welt any wax and aid complete flattening of tissue
▪ Manipulate the ribbon so that the tissue is in the centre of the slide
▪ Remove any debris as necessary
▪ Label slides with name of scientist, date, lab number, microtome number
▪ Compare slide to block to ensure full face achieved
Double check labelling

Question 13

What is done in the collecting and drying step

Answer 13

○ If for H&E then they can be racked and placed on automated stainer
○ Spares for special staining should be placed in the 65 degrees incubater for an hour to melt sections onto the slide
○ Spares should be collected and filed in the SPARES RACK
○ Trucut Breast biopsies, trans rectal prostatic biopsies and IHC spares should all be collected separately and labelled according
IHC slides should be placed in the 60 degrees incubater in the IHC department for an hour to allow the melting of sections onto the slides

Question 14

What is done in the ID step

Answer 14

Scientist needs to initial all the slides they’ve made as well as record the microtome number so that all areas of the process are traceable

Question 15

What tissues may need to be slightly warmed up before microtomy

Answer 15

• Haemorrhagic tissue may need to be warmed up (handling tissue/pressing thumb to tissue face is usually enough)
  Tissue can also be softened in water

Question 16

How would you know if a tissue has not processed correctly

Answer 16

• Middle of the black has a difference consistency – softer – may even be wet
  May see semi-processed blood on blade

Question 17

What should you do if a tissue is inadequately processed

Answer 17

• Usually is due to insufficient fixation in 10% formalin
  
  • If large specimen -> can go back for more tissue and process agains from the start -> additional time in fixative
    If no more tissue I.e. small tissue, biopsy, area of interest etc then block will have to be melted down – reprocessed -> brought back to formalin and given sufficient additional time in formalin

Question 18

What should you do if a tissue is insufficiently decalcified

Answer 18

• Slowly and carefully trim the until the calcified area is exposed
  
  • Place the block in RDC solution for only a few minutes
  • Remove from RDC and place in a bucket of cold tap water – wash thoroughly
  • Place block onto cold plate and attempt to cut a representative section again
  • Can repeat if needed
    If surface decalcification not sufficient then melt block and reprocess -> consult senior first as staining properties of tissue will be affected

Question 19

When floating your sections what does the alcohol bath do?

Answer 19

▪ Alcohol reduces surface tension which allows ribbon to flatten out with ease particularly when transferred to heated water bath

Question 20

When floating your sections what does the warm water bath do?

Answer 20

▪ The warm water bath will welt any wax and aid complete flattening of tissue

Question 21

What should be done to slides that require special stains?

Answer 21

○ Spares for special staining should be placed in the 65 degrees incubater for an hour to melt sections onto the slide

Question 22

What should be done to slides that require IHC?

Answer 22

IHC slides should be placed in the 60 degrees incubater in the IHC department for an hour to allow the melting of sections onto the slides