EXERCISE 4B: TRIMMING AND CUTTING OF PARAFFIN SECTIONS

ROUTINE TISSUE PROCESSING

TRIMMING

Trimming is also known as __________

“facing”, or “roughing”.

TRIMMING

The __________ is removed from the mold and the ________ is noted.

NUMBER 1

solidified paraffin tissue block

accession number

TRIMMING

The ________ is trimmed down with a scalpel blade so that the tissue block can fit the ________.

Please note that all sides must be ___________.

The __________ of the tissue block are trimmed until perfectly level and all sides are ________, almost to the edge of the tissue.

NUMBER 2

sides of the paraffin block

block holder of the microtome

parallel to each other

sides, top and bottom

parallel

TRIMMING

____________ is removed from the block to partly expose the issue surface.

The goal of properly trimming a block is to conservatively _____________.

NUMBER 3

Excess paraffin wax

expose the tissue down to a level where a representative section can be obtained

TRIMMING

Trimming away excess paraffin wax can produce ________, allowing you to place multiple sections in a single slide.

shorter ribbons

MICROTOMY

PREPARATION

Arrange the blocks in a ____________ to give both the tissue and the paraffin similar consistency.

Sectioning is generally improved when the specimen and the wax are well matched in _________. It is for this reason that most paraffin blocks must be _______ when sections are cut. The actual method used to chill the block is important.

numerical order on a cooling device

hardness, cold

MICROTOMY

PREPARATION

________________ provides better support for the harder elements in a specimen allowing thinner sections to be obtained.

Cold wax

MICROTOMY

PREPARATION

The blocks are placed on a ___________________ for a few minutes (surface of melting ice).

cold plate or a cold wet surface

MICROTOMY

PREPARATION

__________ penetrates a small distance into the block face, ______________ and making them more amenable to cutting. This is particularly important to ___________________.

Water

swelling the tissues

over- dehydrated, dry or crumbly tissues

MICROTOMY

PREPARATION

PREPARATION

PREPARATION OF THE WATER BATH

What temperature?

Paraffin wax shrinks up to ________, causing compression in tissues. However, this compression is almost fully recovered when sections are _________

40°C - 45°C (5°-10°C lower than the melting point of paraffin)

15% upon cooling down

floated on a warm floatation bath or water bath

CLAMPING THE SPECIMEN

Orient the _________. The ___________ can affect the ease with which a ribbon can be obtained and directly influencesection quality.

cassette/block in the block holder

orientation of the specimen to the blade during the cutting stroke

CLAMPING THE SPECIMEN

Blade passes through the mucosa last.

Intestine

CLAMPING THE SPECIMEN

It is better to present a point of dense tissue to the blade rather than a straight edge.

Cervix

CLAMPING THE SPECIMEN

Blade passes through the epidermis last.

Skin

CLAMPING THE BLADE

Modern microtomes make use of ________. These are locked onto the ________.

disposable blades

knife holder

CLAMPING THE BLADE

The _______ protects the user from getting cut.

knife guard handle

CLAMPING THE BLADE

Lift it _____ when manipulating the tissue block.

up

CLAMPING THE BLADE

Lift it _____ to reveal the blade and start cutting.

down

CLAMPING THE BLADE

Insert the ________ and tighten to lock in place.

_______ are present. The ________ secures the blade itself, while the ________ secures on the knife/blade holder.

Both clamps must be in this position to properly section.

knife blade into the holder

Two clamps

Right clamp, left clamp

ADVANCE THE BLOCK TO THE KNIFE

Generally, you bring the knife guard _________ (dependent on the model of microtome being used).

Some microtomes have knife guards that can be ________.

closer to the block, not the other way around

manually adjustable

ADVANCE THE BLOCK TO THE KNIFE

__________ can cause vibrations during sectioning, and produce _________.

Extending the sample holder/block holder

chattering artifacts in the slide

CHATTERING ARTIFACTS

________, most often results from ________ during processing, but may also result from a __________.

____________ will help correct this problem if it is caused by excessive dehydration of the tissue.

Chatter, or microvibration

overdehydration

dull blade, too much blade tilt, or cutting too rapidly

Soaking the faced block with moistened cotton

ADVANCE THE BLOCK TO THE KNIFE

To avoid chatter, move the block holder backwards using the ____________

coarse feed wheel

ADJUST MICROMETER

Select the appropriate tissue section thickness by ___________. Routine sections are cut at _________.

Other factors such as ___________ can influence the actual thickness achieved.

adjusting the thickness scale/ micrometer gauge

4-6um

speed of rotation, clearance angle setting and the condition of the cutting edge

ADJUST MICROMETER

The thickness of the section can be adjusted ________ , but be aware to avoid _________ to avoid thick sections.

based on the tissue specimen

higher settings (>10um)

ADJUST CLEARANCE ANGLE

While the thickness gauge sets thickness of the section cut, the ______ sets the clearance angle. _______ must be set for _________.

Set the correct clearance angle required which is ______

knife holder

Clearance angle, optimum performance

0 - 15°

ADJUST CLEARANCE ANGLE - ERROR.

This most frequently results in sections that are missed or skipped, alternately thick and thin, wrinkled and jammed, or lifted from the blade.

The clearance angle, or blade tilt, is too slight

ADJUST CLEARANCE ANGLE - ERRORS

This frequently causes chatter, microvibration, washboarding, or undulations in sections and may make it impossible to obtain a ribbon.

The clearance angle, or blade tilt, is too great.

CUTTING POSITION

Bring the specimen on the cutting position by turning the ________.

hand wheel

CUTTING POSITION

Hard tissues cut best with _________.

firm, relatively rapid stroke

CUTTING POSITION

Soft tissues cut best with _______.

* Make sure that the blade is _______ to the tissue block.

slow gentle motion

parallel

CUTTING POSITION

The entire face of the block was ______ to the blade, so that ribboning was very uneven, and a _________.

not parallel

major part of one side of the block has been cut away

CUTTING POSITION

The block is _______ to the blade edge. The top and bottom edges of the block should be _______, and the bottom of the block should be ______ to the blade edge, or crooked ribbons and ___________ may result.

not parallel

parallel

parallel

poor adherence of one section to the next

SECTIONING PROPER

As sections are cut, a _____ is created because successive sections stick edge-to-edge due to local pressure with each stroke.

To ensure proper sectioning and a good ribbon, the wheel should be turned __________.

ribbon

clockwise, at a moderate and even phase

PULLING RIBBON

The ribbon will roll _________ the face plate of the knife holder. As the ribbon comes off the knife, the loose edge of the ribbon is held with __________. The other end of the ribbon is then lifted from the knife using _________. Care must be taken to _________ to avoid breaking it.

down

a pair of forceps

forceps or a wooden applicator stick

not pull the ribbons taut

SET RIBBONS ASIDE

A length of about _____ is sufficient to detach the tissue ribbon from the microtome. This is around ________ to allow easy location of serial sections.

Set aside ribbons of paraffin sections in a ________.

You may also float them immediately into the __________.

6 inches

ribbons of ten

wooden tray with their accession numbers

water bath to continue processing

TROUBLESHOOTING: MICROTOMY

COMPRESSED, WRINKLED, OR JAMED SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Use of dull knife/blade edge

Resharpen the knife or move the existing blade to an unused surface or replace the blade with a new one.

TROUBLESHOOTING: MICROTOMY

COMPRESSED, WRINKLED, OR JAMED SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Paraffin sticking in the back side of the holder

Keep the edge free of paraffin by wiping (up, never down) with gauze slightly dampened in xylene. Remove excess xylene with another gauze soaked in absolute alcohol.

TROUBLESHOOTING: MICROTOMY

COMPRESSED, WRINKLED, OR JAMED SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Unparalleled block edges

Trim the block edges until they are parallel.

TROUBLESHOOTING: MICROTOMY

COMPRESSED, WRINKLED, OR JAMED SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Too warm room

Cool/change the room temperature or cool the block in ice water

TROUBLESHOOTING: MICROTOMY

COMPRESSED, WRINKLED, OR JAMED SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Too little knife/blade tilt

Increase the tilt of the blade (greater clearance angle).

TROUBLESHOOTING: MICROTOMY

COMPRESSED, WRINKLED, OR JAMED SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Too rapid cutting

Decrease cutting speed (1 revolution per second) for very thin sections.

TROUBLESHOOTING: MICROTOMY

SKIPPING OF SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Very large blocks or blocks with hard regions that may damage the spring knife/blade edge while sectioning or materials not properly hardened

Specimen needs to be treated by softening the block:

• Chill the block further

• Soak the block in a bowl of soapy water

TROUBLESHOOTING: MICROTOMY

SKIPPING OF SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Too much tilting of the knife/ blade which scrapes off the section instead of cutting it

Adjust the knife/blade so that the clearance angle between the cutting facet of the knife/blade and specimen is correct.

TROUBLESHOOTING: MICROTOMY

SKIPPING OF SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Too little tilting of the knife/blade resulting in compression of the block on the return stroke.

Increase the tilt of the blade (greater clearance angle)

TROUBLESHOOTING: MICROTOMY

SKIPPING OF SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Worn-out microtome due to lack of lubrication and not in adjustment

Ensure that the microtome is in good working order and that routine maintenance is scheduled and documented. Have the microtome checked by the manufacturer.

TROUBLESHOOTING: MICROTOMY

SKIPPING OF SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Loose clamping set screws

Retighten screws and clamp firmly.

TROUBLESHOOTING: MICROTOMY

SKIPPING OF SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Paraffin too soft for tissue

Remove excess paraffin from the edge of the block

TROUBLESHOOTING: MICROTOMY

SKIPPING OF SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Use of dull knife/blade

Re-sharpen the knife or move existing blade to an unused surface or replace blade with a new one.

TROUBLESHOOTING: MICROTOMY

GROOVED, SCORED, SMEARED, AND DEFORMED SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Defect or nicks in the knife/ blade edge

Move the blade to an unused area or change the blade/knife, then cut the block. Change the blade or re-sharpen the knife.

TROUBLESHOOTING: MICROTOMY

GROOVED, SCORED, SMEARED, AND DEFORMED SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Dirt/calcium, bone or other hard particle/ material present in the block

• Check the tissue for foreign bodies, staples, calcium, etc. If present, remove carefully to avoid damaging the tissue.

• (Note: If the defect remains in exactly the same area of the new section, then the problem is the specimen).

• Refilter dirt in paraffin.

• Decalcify the tissue.

TROUBLESHOOTING: MICROTOMY

SCRATCHES, LINES, AND SLITS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Too much knife/ blade tilt

Decrease tilt of the knife/blade so it will cut rather than scrape.

TROUBLESHOOTING: MICROTOMY

SCRATCHES, LINES, AND SLITS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Dirty stock solution

Filter the stock solution

TROUBLESHOOTING: MICROTOMY

SCRATCHES, LINES, AND SLITS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Dirty knife/blade edge

Clean the knife/blade edge

TROUBLESHOOTING: MICROTOMY

SCRATCHES, LINES, AND SLITS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Tissue too hard for paraffin

Use celloidin-embedding materials or soak in water to soften

TROUBLESHOOTING: MICROTOMY

SCRATCHES, LINES, AND SLITS

_____________________ can also occur when an undamaged knife becomes damaged due to contact with hard tissue. Thus, ____________________ (applying on the tissue block) may be recommended while cutting.

Knife lines or scoring

surface decalcification

TROUBLESHOOTING: MICROTOMY

BRITTLE AND SHATTERED SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Incomplete dehydration or clearing

Rehydrate the block

TROUBLESHOOTING: MICROTOMY

BRITTLE AND SHATTERED SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Incomplete infiltration of soft and mushy tissue with embedding material

Re-infiltrate tissues with paraffin and reembed.

TROUBLESHOOTING: MICROTOMY

BRITTLE AND SHATTERED SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Too long in paraffin bath or too hot bath

Try chloroform or toluene instead of xylene, or a mixture of toluene and cedar oil as clearing agents.

TROUBLESHOOTING: MICROTOMY

BRITTLE AND SHATTERED SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Too long in paraffin bath or too hot bath

Try chloroform or toluene instead of xylene, or a mixture of toluene and cedar oil as clearing agents.

TROUBLESHOOTING: MICROTOMY

LIFITNG IF SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Use of a dull knife/blade edge

Resharpen the knife or move the existing blade to an unused surface or replace the blade with a new one.

TROUBLESHOOTING: MICROTOMY

LIFITNG IF SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Too little knife/blade tilt

Increase the tilt of the blade (greater clearance angle)

TROUBLESHOOTING: MICROTOMY

LIFITNG IF SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Warm room and paraffin that is too soft

a. Change to a harder paraffin (slightly higher melting point).

b. Cool the room or cool the block in ice water.

TROUBLESHOOTING: MICROTOMY

PLEATED, FOLDED, CROOKED RIBBONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Imperfections in the blade edge, i.e., the horizontal edges (top and bottom) are not parallel or the lower block edge is not parallel to the knife edge when sectioning

a. Trim the block parallel to the other edge or reembed them in molds so that the edge of block is parallel to the knife edge.

b. Try another part of the knife edge.

TROUBLESHOOTING: MICROTOMY

PLEATED, FOLDED, CROOKED RIBBONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

The block is not evenly chilled or the hardness of the paraffin varies from one side of the block to the other

Ensure that the block is evenly chilled and hardened.

TROUBLESHOOTING: MICROTOMY

PLEATED, FOLDED, CROOKED RIBBONS

_____________ can typically occur in specimen such as cartilage tissue, unless using specialized techniques (doubleembedding, resinembedding)

FOLDING

TROUBLESHOOTING: MICROTOMY

FAILURE TO FORM RIBBONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Block holder not parallel to the blade causing too much shaving of the block on one side.

At the start of sectioning, adjust the block holder so that the block face and the blade are perfectly parallel.

TROUBLESHOOTING: MICROTOMY

FAILURE TO FORM RIBBONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Uneven block margins

Re-trim the tissue block to become parallel

TROUBLESHOOTING: MICROTOMY

FAILURE TO FORM RIBBONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Dull blade

Re-sharpen or replace blade

TROUBLESHOOTING: MICROTOMY

FAILURE TO FORM RIBBONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Paraffin is too sticky, causing sections to stick to the knife guard

Try chilling the block first before cutting

TROUBLESHOOTING: MICROTOMY

HOLES IN THE SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Block positioned too aggressively

Position the block less aggressively with smaller micrometer advances of the block for each removed section

TROUBLESHOOTING: MICROTOMY

HOLES IN THE SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Small flecks of tissue removed from the block

If there is sufficient tissue in the block, cut and discard ribbons until the holes disappear

TROUBLESHOOTING: MICROTOMY

HOLES IN THE SECTIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Excessive dehydration or improperly processed tissue

Expose the tissue, then soak the block in ice water or with a wet piece of cotton before sectioning

TROUBLESHOOTING: MICROTOMY

This effect may occur when sectioning lymph node and other very cellular organs.

Excessively rough trimming pulls tissue fragments from the block face and these appear as ____________________.

HOLES IN THE SECTIONS

holes in subsequent thin sections

TROUBLESHOOTING: MICROTOMY

WASHBOARDING / UNDULATIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Worn microtome pants that allow too much tolerance in some of the moving parts

This commonly occurs in very hard tissues such as the uterus, or in overfixed tissues. This is easily seen when sections are floating on top of the water bath.

TROUBLESHOOTING: MICROTOMY

WASHBOARDING / UNDULATIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Loose clamping of the blade or block

Make certain that the microtome is in good working condition and is routinely scheduled for maintenance.

TROUBLESHOOTING: MICROTOMY

WASHBOARDING / UNDULATIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Loose screws

Tighten all screws. Ensure that the block holder shaft is not overextended.

TROUBLESHOOTING: MICROTOMY

WASHBOARDING / UNDULATIONS

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Loose knife holder

Clamp tightly. The block and blade should be tightly clamped in the microtome.

TROUBLESHOOTING: MICROTOMY

WASHBOARDING / UNDULATIONS

Also known as ____________________.

coarse chatters

TROUBLESHOOTING: MICROTOMY

Often occurs in cutting dense tissue such as uterus and cervix

WASHBOARDING / UNDULATIONS

TROUBLESHOOTING: MICROTOMY

CHATTER

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Over-hydration or lack of moisture in tissue

Restore the moisture of the tissue by letting the block stand and soaking it briefly in ice water or by placing it face down on an ice tray

TROUBLESHOOTING: MICROTOMY

CHATTER

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Use of dull knife/ blade

Resharpen the knife/blade, move the existing knife/blade for an unused surface, or replace it with new blade

TROUBLESHOOTING: MICROTOMY

CHATTER

PROVIDE THE TROUBLESHOOTING TECHNIQUES

Too much blade tilt or cutting too quickly

a. Decrease the tilt of the blade (smaller clearance angle).

b. Decrease the cutting speed (1 revolution per second)

TROUBLESHOOTING: MICROTOMY

CHATTER

__________________ are fine parallel cracks usually caused by tiny vibrations in the knife edge as it passes through hard, brittle blocks.

Chatter or venetian blinds

TROUBLESHOOTING: MICROTOMY

OTHER PROBLEMS

Note the hole in the middle of the tissue section.

Poorly processed liver that will not produce a cohesive section

TROUBLESHOOTING: MICROTOMY

OTHER PROBLEMS

The tissue is not completely dehydrated and/or cleared and is therefore not properly infiltrated (the wax could not penetrate due to the presence of water). Reprocessing should help.

Indicates under-processing.

TROUBLESHOOTING: MICROTOMY

OTHER PROBLEMS

SECTION IS TOO THICK

 Wrong micrometer setting

 Sectioning at too great a speed

 Microtome needs recalibration

TROUBLESHOOTING: MICROTOMY

TISSUE COMPRESSION, NICKS

Compressed sections, as seen in this figure, may result from a _______________________.

dull blade, too little blade tilt, paraffin accumulation on the blade, and cutting too rapidly

TROUBLESHOOTING: MICROTOMY

TISSUE COMPRESSION, NICKS

The results of a _____________ can be seen in this paraffin ribbon. This defect possibly resulted from improper handling of the forceps used in sectioning. Any ______________ can damage the edge and cause this type of artifact.

major nick, or defect, in the blade edge

metallic object that is allowed to come into contact with the blade edge

MICROTOMY

PROCESS OF MICROTOMY

1. PREPARATION

2. CLAMPING THE SPECIMEN

3. CLAMPING THE BLADE

4. ADVANCE THE BLOCK TO THE KNIFE

5. ADJUST MICROMETER

6. ADJUST CLEARANCE ANGLE

7. CUTTING POSITION

8. SECTIONING PROPER

9. PULLING RIBBON

10. SET RIBBONS ASIDE

TROUBLESHOOTING: MICROTOMY

1. Compressed, Wrinkled, or Jammed Sections

2. SKIPPING OF SECTIONS

3. Grooved, Scored, Smeared, and Deformed Sections

4. Scratches, Lines, and Splits

5. Brittle and Shattered Sections

6. LIFTING OF SECTIONS

7. PLEATED, FOLDED, CROOKED RIBBONS

8. FAILURE TO FORM RIBBONS

9. HOLES IN THE SECTIONS

10. WASHBOARDING / UNDULATIONS

11. CHATTER

12. OTHER PROBLEMS

13. SECTION IS TOO THICK

14. TISSUE COMPRESSION, NICKS