350 - Topic 7 (Microtomy) Flashcards
routine paraffin blocks are cut at…
4-5 um
exceptions to 4-5 um rule
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
the microtomist’s choice of blade is influenced by …
- embedding media used
- style of microtome
these large steel blades require frequent re-sharpening
wedge blades
- blades edge must be examined microscopically to ensure it is properly honed prior to sectioning
T or F. disposable steel blades are not hard or sharp enough to cut resin-embedded blocks
T
what do techs use to cut resin-embedded blocks?
glass or diamond blades
- used to cut very hard tissues or very thin (<1um) sections
glass vs diamond blades
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.)
this is used to cut ultrathin sections for light or electron microscopy (<0.5 um)
ultramicrotomes
microtome setup
- 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
the angle formed between the edge of the blade and the surface of the block
clearance angle
ideal clearance angle
position the blade so that the bevel face is parallel with the path of motion of the block
how can most sectioning issues be corrected?
combination of chilling and soaking ASSUMING that tissue has been properly processed
if serial sections or steps/levels are required, the block may require additional…
chilling and soaking between cuts
facing
- 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
chilling
- aka cooling or soaking
- blocks must be cooled prior to fine sectioning
- cooling hardens wax = thin, artifact-free sections
- too warm = compressed sections
sectioning
- aka cutting
- after at least 15 mins of cooling
- one rotation per second
levels
- 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
serial sections
- 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
routine sections
- aka random sections
- single section collected and mounted on the slide
- used for cases where there are many blocks of tissue
- organ resections
deepers/recut
- 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
factors affecting microtomy (prior to fine cutting)
- 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
factors affecting microtomy (during fine cutting)
- 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
how long are slides placed in a drying oven for and at what temp?
- 30 mins to an hour
- no hotter than 60C
- may be dried at 37C overnight for superior cellular detail
disadvantages to using microwaves to dry slides
possibility of ‘cooking’ areas of the tissue
water and fat both attract more microwaves than other tissue components
common misconception of drying slides in oven
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)
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
adhesives or charged slides
ex: silver stains (alk solutions), epitope retrieval methods for IHC (heat, moisture, enzymes)
when would a tech use a charged slide for standard staining procedure
when sectioning tissues are friable (bloody tissues)
adhesive slides
- added to slides or directly to water bath
- gelatin, albumin, Elmer’s glue, poly-L-lysine
major disadvantage of adhesives
their tendency to produce background staining (when applied in excess or when not fully dissolved in water bath)
T or F. most sections have a net positive charge and are attracted to charged slides
F! sections have net neg charge
slides = pos charge
adhesives may be applied to slides or added to water bath to prevent section loss; this is esp. important for …
thick sections (>7um)
staining methods have high pH reagents
