EMBEDDING & MICROTOMY

How do you embed tubular structures?

(Fallopian tubes, appendix) embed in cross section so that the lumen (hole in the middle) and all layers of the tubular wall (mucosa, serosa and inner wall) can be seen microscopically.

Explain ergonomics of waterbath placement

*adjust your waterbath so you can see the whole thing in proper lighting *place it on the left or right side depending on your dominant hand *place it close enough to you so that you don't have to twist or stretch to reach it

Explain microtome ergonomics

*bring your microtome to the edge of the table so that you can see straight down on the knife holder *adjust your chair to keep your hands at the knife blade level so you can relax your shoulders and not hold them up *adjust the back of your chair to support your back as needed

How do you acheive complete adhesion?

*fill the mold with melted paraffin up over the well *do not allow the surface of the paraffin to start to solidify *lay your cassette at an angle to avoid bubbles *be sure to fill your cassette to the top!

Explain the rocking technique

*look at the face with each swipe of the blade *bring the block down against the blade with your right hand *adjust the thickness of each swipe with your left hand

How do you know your slide is dry?

*no water left on slide *tissue should be transparent when it comes out *paraffin should be melted *tissue should not have any bubbles *if it's not dry enough, the section will wash off during staining

Explain Microtome Maintenance

*remove the blade first!!! *clean your waterbath and microtome after each use! *sweep all loose paraffin into the garbage using a paint brush *take the microtome apart and get all of the paraffin off of it

Explain how to setup for cutting

*return the chuck all the way back towards the microtome *position the knife holder. *adjust the microns *adjust the knife angle *make sure all components are tight *insert the blade

How do you clean the embedder?

*wipe all surfaces clean *fill paraffin well *clean forceps holders with Qtip *clean molds if necessary *turn off cold plate, not the heat or your paraffin will solidify! *clean out small cracks on hot plate with Qtip *clean paraffin trap/tray

Name four more things about the consequences of poor orientation.

1. If the specimen is not salvageable then it could be destroyed! 2. If the specimen is cut through, patient is re-biopsied at the cost of your pathologist! 3. All of the tumor could have existed in the first specimen! 4. You could potentially cost a patient their lives!

Give four reasons why a specimen should be re-embedded.

1. One corner is lifted. 2. Improper alignment. 3. Not on same plane. 4. General embedding orientation is incorrect.

If the orientation is not correct then........... (3 things)

1. Specimen gets cut incorrectly 2. Specimen is stained and given to the pathologist and they cannot interpret the slide. 3. Delay in patient results IF the specimen is salvageable!

What will the log tell you? (6)

1. case number 2. number of cassettes per case 3. sub numbers used 4. number of specimens in each cassette 5. Tissue type (for orientation) 6. Any special instructions

Name the areas of the embedding center (10)

1. large cold plate 2. hot well 3. temp display 4. paraffin dispenser 5. small cooling plate 6. cassette warmer 7. paraffin well 8. mold warmer 9. hot plate 10. paraffin waste catch

Distinguished Orientation: How will you know which way to put the specimen? (3 things)

1. notching 2. inking 3. instructions on side of the cassette

Name the QA/QC standards for embedding

1. side that goes down in the cassette goes down in the mold. 2. distinguished orientation: notching, inking, side of the cassette 3. Apply slight pressure to ensure tissue is on the same plane. 4. Embed tissues in the center of the mold so that none of the tissue is touching the edges. 5. Wipe forceps between cases to avoid cross contamination. 6. Clean forceps holders daily. 7. Match the slide to the block to ensure proper orientation and cutting has occurred. 8. Use M for multiple pieces or N for numerous. 9. Utilize and follow the log!

What should the wedge angle be at?

15-18 degrees

What should the bevel angle be at?

27-32 degrees

What should the clearance angle be at?

3-8 degrees

How many blocks per hour are standard for embedding?

40-60 blocks per hour.

What does adhesion mean?

Adhesion is the surface of the cassette and the mold casing attaching together.

How do you embed hard tissues?

At a slight angle to the knife, so that the tissues won't pop out of the block.

What do you do once you have your slides?

Bake them in a metal rack and put them in the oven at 60* C for 30 minutes or 110* C for 15 minutes

______________ is the angle of the blade itself.

Bevel

What is happening when you have length wise scratches or splits?

Defective knife, paraffin build up on knife, hard objects in the block such as staples

QA/QC Standards: Side ______________ in the cassette is the side that goes _________________ in the mold.

Down, down

How do you determine the wedge angle?

Draw an imaginary line from the lower portion of the bevel angle or edge and the intersecting point is the wedge angle.

How do you clean your waterbath?

Dump out the water, clean off all of the paraffin, bleach weekly with 10% bleach for 10 minutes.

What does facing mean?

Facing is the act of shaving off the face of the block to achieve a full cut of the tissue present.

What do you need to do to your slide after picking it up out of the water bath?

Flick the water off of it!

What kind of knives are used for cutting plastic embedded tissues?

Glass knives

How do you embed multiple small tissues?

In a line parallel or slanted in the block. Never place them randomly. This makes the pathologist hunt for the biopsy potentially missing one during microscopic evaluation.

Where should tissues be placed in the mold and why?

In the center of the mold so that no tissue is touching the edge of the mold.

What is wrong when your sections attach to the top of the block?

Knife is dull or knife tilt is too slight

What can poor orientation make very difficult?

Microtomy

Utilize M on the side of the block. What does this stand for?

Multiple pieces were in the block.

What is happening when you have chatter?

Over dehydration, (soak your blocks), improper knife angle

What is happening when you have parched earth?

Over dehydration, water bath too hot

Which microtome is used most commonly?

Rotary

What happens when your clearance angle is too slight?

Skipped sections

What type of microtome are you using if the block is stationary?

Sliding

What type of microtome moves the knife horizontally across the block?

Sliding

Which microtome is most commonly used with celloidin and large tissue blocks?

Sliding

What are the 5 dry tissues?

Spleen, tonsil, lymph node, thyroid, liver

What is the bevel angle?

The actual angle of the tip of the knife or knife edge.

What is wrong when you have chopping into the block?

The knife is not clamped down, block is incorrectly positioned in the chuck, too aggressive facing, too fast fine feed, hard tissue

What is happening when you have skipped sections/thick thin sections?

Too little of a knife tilt, loose or worn microtome parts (improper maintenance)

_________________ is an imaginary line of each bevel edge to intersection.

Wedge

What is wrong when you have holes in the section?

aggressive facing

What happens when your clearance angle is too great?

chatter micro vibration of the knife, washboarding

What angle is the only one that you can adjust?

clearance angle

What kind of microtome has a freezing chamber to reduce airborne disease?

cryostat

What kind of microtome is contained?

cryostat

What microtome cuts like a rotary?

cryostat

What microtome is used for freezing "stat" tissues?

cryostat

_______________ should be ________________ between ______________ to avoid __________ __________________________.

forceps, wiped, cases, cross contamination

Which microtome clamps to a table top?

freezing

Which microtome is considered portable?

freezing

Which microtome uses carbon dioxide to freeze the specimen?

freezing

Which microtomes have been replaced by cryostats?

freezing

How do you embed tissues with walls?

gallbladder, GI tract, should be embedded on edge with all the layers of the wall visible and all layers are facing the same direction.

What is happening when you have washboarding?

hard tissues, block or knife not tightly clamped down

What is happening when you have compressed sections?

knife is dull, knife is gummed up with paraffin, paraffin build up on the back of the knife, too rapid of cutting speed, block too warm

Utilize and follow the _____________!

log

_____________ the ___________ with the _____________ to determine if proper orientation and cutting has occurred.

match, slide, block

What is wrong when you have knife lines?

nick in the knife, move or discard knife

What is wrong when your ribbons are crooked?

poor adherence of section to the next, top and bottom edge of the block is not parallel to the knife

Name the three diffferent types of microtomes

rotary, sliding, freezing

QA/QC standards: _______________ _________________: applied to ensure the tissue is flat or that all tissues are _______ ________ _____________ ______________.

slight pressure, on the same plane

What is happening when your sections are flying away?

static electricity or drafts, not hanging on to that ribbon

What kind of knives are used for paraffin sectioning?

steel knives

What is happening when you're chucking your block?

too high of a clearance angle

What does a scraping sound on the microtome mean?

too little of clearance

How do you embed skin?

with the epithelium/epidermis facing one direction.

What do you do immediately after facing the block?

you must get rid of the aggressive face by taking two to three 4 micron swipes at the block to get rid of the holes in your section