350 - Topic 7 (Microtomy)

Question 1

routine paraffin blocks are cut at…

Answer 1

4-5 um

Question 2

exceptions to 4-5 um rule

Answer 2

very cellular tissue (lymph nodes and kidney biopsies); may be cut at 2-3 um
less dense tissue (breast, brain) may be cut thicker (6-8um)

some special testing procedures require thinner or thicker sections as well

Question 3

the microtomist’s choice of blade is influenced by …

Answer 3

• embedding media used
• style of microtome

Question 4

these large steel blades require frequent re-sharpening

Answer 4

wedge blades

• blades edge must be examined microscopically to ensure it is properly honed prior to sectioning

Question 5

T or F. disposable steel blades are not hard or sharp enough to cut resin-embedded blocks

Answer 5

T

Question 6

what do techs use to cut resin-embedded blocks?

Answer 6

glass or diamond blades
- used to cut very hard tissues or very thin (<1um) sections

Question 7

glass vs diamond blades

Answer 7

glass = inexpensive; quickly lose their edge but extremely sharp; but require frequent replacement

diamond = more expensive but ‘never’ lose their edge; suitable for cutting even the hardest tissue (calcified bone for ex.)

Question 8

this is used to cut ultrathin sections for light or electron microscopy (<0.5 um)

Answer 8

ultramicrotomes

Question 9

microtome setup

Answer 9

• check angle of universal cassette clamp (should be parallel with back of knife holder)
• check clearance angle (3-8 degrees; adjust before facing)
• ensure all levers are firmly tightened
• retract specimen advance mechanism (‘neck’) sing coarse drive wheel
• insert new blade into knife holder

Question 10

the angle formed between the edge of the blade and the surface of the block

Answer 10

clearance angle

Question 11

ideal clearance angle

Answer 11

position the blade so that the bevel face is parallel with the path of motion of the block

Question 12

how can most sectioning issues be corrected?

Answer 12

combination of chilling and soaking ASSUMING that tissue has been properly processed

Question 13

if serial sections or steps/levels are required, the block may require additional…

Answer 13

chilling and soaking between cuts

Question 14

facing

Answer 14

• aka paring
• trimming away excess wax
• expose full face of tissue
• proper embedding and orientation is critical to getting a full face without paring through sample

Question 15

chilling

Answer 15

• aka cooling or soaking
• blocks must be cooled prior to fine sectioning
• cooling hardens wax = thin, artifact-free sections
• too warm = compressed sections

Question 16

sectioning

Answer 16

• aka cutting
• after at least 15 mins of cooling
• one rotation per second

Question 17

levels

Answer 17

• aka step sections
• once a section has been produced, block is trimmed 60-100 um, and a second collection is collected
• process may be repeated
• slides should indicate the depth of section
  (l1 = superficial; l2 = deeper; l3 = deeper than 2, etc.)
• additional chilling between levels may be required

Question 18

serial sections

Answer 18

• most challenging
• sections collected without any steps or levels
• every section in the ribbon is mounted on a slide
• important not to skip any sections!!
• slides labeled accordingly
• small biopsies

Question 19

routine sections

Answer 19

• aka random sections
• single section collected and mounted on the slide
• used for cases where there are many blocks of tissue
• organ resections

Question 20

deepers/recut

Answer 20

• when pathologists need to see additional slides from deeper in the block to render a diagnosis
• further sections taken after chilling; according to levels
• standard number of slides when request deeper slides= usually 2 or 4

Question 21

factors affecting microtomy (prior to fine cutting)

Answer 21

• fixation: ensures relationship between cells and extracell components is maintained during processing
• processing: tissue must be fully infiltrated w wax to adequately support tissue during microtomy
• microtome: clean, well-maintained, with all locking levers tightened
• temperature: blocks must be cold for thin sections’ tissue softened (water) to facilitate sectioning

Question 22

factors affecting microtomy (during fine cutting)

Answer 22

• cutting speed: use even, gentle pressure on handwheel and cut at about 1 rotation per second
• ribbons: shorter ribbons easier to manipulate; handle with applicator sticks or forceps to prevent injury
• waterbath: check temp daily (about 10C below MP of wax) and skim after every block
• section retrieval: once section free of wrinkles, carefully pick up onto slide and let dry vertically

Question 23

how long are slides placed in a drying oven for and at what temp?

Answer 23

• 30 mins to an hour
• no hotter than 60C
• may be dried at 37C overnight for superior cellular detail

Question 24

disadvantages to using microwaves to dry slides

Answer 24

possibility of ‘cooking’ areas of the tissue
water and fat both attract more microwaves than other tissue components

Question 25

common misconception of drying slides in oven

Answer 25

that it will remove excess wax from section
yes, it will melt some off but main purpose is to remove any water from section
deparaffinization = xylene (BUT if water remains during this step, it will be incomplete)

Question 26

these are used when slides will be exposed to harsh solutions or pretreatments during staining which could otherwise damage or cause the section to detach

Answer 26

adhesives or charged slides

ex: silver stains (alk solutions), epitope retrieval methods for IHC (heat, moisture, enzymes)

Question 27

when would a tech use a charged slide for standard staining procedure

Answer 27

when sectioning tissues are friable (bloody tissues)

Question 28

adhesive slides

Answer 28

• added to slides or directly to water bath
• gelatin, albumin, Elmer’s glue, poly-L-lysine

Question 29

major disadvantage of adhesives

Answer 29

their tendency to produce background staining (when applied in excess or when not fully dissolved in water bath)

Question 30

T or F. most sections have a net positive charge and are attracted to charged slides

Answer 30

F! sections have net neg charge
slides = pos charge

Question 31

adhesives may be applied to slides or added to water bath to prevent section loss; this is esp. important for …

Answer 31

thick sections (>7um)
staining methods have high pH reagents