Faults and Reason Histopath, Faults and Remedy Histopath

Sections expand or disintegrate on water bath

Poor impregnation of tissue

Water temperature too high in

flotation bath

Sections roll into a coil instead of remaining flat on knife edge

Blade dull

Rake angle too small

Section too thick

Grooved, Scored, smeared and deformed sections

Dull knife/blade edge

Too warm room

Sections fail to form ribbons

Surfaces and edges of the block are not parallel

Horizontal surface of the block is not parallel to the knife

Paraffin wax is too hard

Knife is tilted too much

Sections are too thick

Knife is dull

Sections roll up on cutting so that they adhere and get broken against the knife edge

Knife is blunt

Tilt of knife is too great

Knife edge is dirty

Ribbon is curved, crooked or uneven instead of straight

Blunt of dull spot on the knife, producing an irregular knife edge

Edges of the block are not parallel but round or wedge shape

Knife is not parallel to the block

Paraffin is impure

Sections are compressed wrinkled or jammed

knife is blunt or dull

Paraffin block is warm and soft

Knife edge is coated with paraffin

Sections are too thin

Microtome set screw is loose

Tilt of knife is too vertical

Too rapid cutting

Too warm room

Unparallel block edges

Sections are squashed (width of each section is less than that of block

Bevel of knife is lost due to incorrect sharpening

A hole is formed in the section

Bubble or dirt formed in the embedding medium

Hard spot in tissue due to calcium

Excessive dehydration or improperly processed tissue

Sections of unequal thickness are produced

Tilt of knife is too great or bevel is not cleared hence object is compressed against the knife edge

Clamp set screw on knife or blockholder is loose

Blocks are too large

Blocks are too hard

Sections adhere to the knife or other parts of the microtome

Static electricity due to low atmospheric humidity

Knife edge is dirty

Knife edge is dull

Tilt of knife is too great

Ribbon is split or lengthwise vertical scratches are seen on sections

Nicks or damage on the knife edge

Dirty embedding

Knife edge is dirty

Tilt of knife is too great

Sections are lifted from the knife on the upstrokes

Knife tilt is too great

Knife is dull

Paraffin is too soft or room

temperature is warm

Resistance is felt on the lower part of the section during cutting

Tilt of knife is too small, paraffin block is therefore compressed against the base of the knife towards the end of stroke

Horizontal or parallel lines or furrows across the section (chatters) are seen, forming thin and thick zones

Knife edge vibrates due to hardness of tissue

Tilt of knife is too great

Overhydration or lack of moisture in

tissue

Section cut is sometimes thin sometimes thick

Knife is blunt

Knife is not clamped properly

Tilt of knife is too great

Knife or block holder is loose

Knife tilt is too small that block is compressed by bevel and section is not cut

Knife makes a hard metallic scrapping or ringing sound on backstroke, when section is cut

Tilt of knife is too slanted or too big

Tissue is too hard

Knife blade is too thin

Frozen tissue crumbles and comes off the block holder when cut

Freezing is not adequate

Frozen tissue chips into fragments when cut

Tissue is frozen too hard

Pleated, folded and crooked ribbons

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

Brittle or Hard tissues

Prolonged fixation

Prolonged dehydration

Prolonged clearing

Prolonged paraffin infiltration

Overheated paraffin oven

Drying out of tissue before actual

fixation

Clearing agent turns milky as soon as tissue is placed in it

Water not completely removed du to incomplete dehydration

On trimming, tissue smells of clearing agent

Clearing agent not completely removed due to insufficient impregnation

Tissue is opaque, section cutting is difficult due to presence of alcohol

Insufficient clearing

Tissue shrinks away from wax when trimmed

Insufficient dehydration therefore

incomplete impregnation

clearing and

Tissue is soft when block is trimmed

Incomplete fixation

Airholes are found on tissue during trimming

Incomplete impregnation

On trimming wax appears crystalline

Contaminated wax

Block not cooled rapidly enough

Paraffin block, after cooling is moist and crumbles

Insufficient paraffin impregnation

Inadequate processing of fatty tissues

Routine schedule and reagents may not process fat adequately

Hard, dense tissues

Tough keratin component of certain tissues

On embedding, tissue is soft and mushy in the center

Inadequate processing due to very thick or fatty sections

Sections expand or disintegrate on water bath

Re-process tissue

Turn down the temperature of

the flotation bath

Sections roll into a coil instead of remaining flat on knife edge

Use a new blade

Reduce blade tilt if clearance

angle is excessive

Reduce section thickness

Grooved, Scored, smeared and deformed sections

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

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

Sections fail to form ribbons

Re-trim the block

Re-adjust and re-orient the

block

Coat horizontal edges of the block with wax of lower melting point

Reduce the tilt

Re-adjust thickness of the

section

Hone and strop

Sections roll up on cutting so that they adhere and get broken against the knife edge

Sharpen the knife

Reduce the tilt

Clean the knife edge

Ribbon is curved, crooked or uneven instead of straight

Adjust the knife so that knife edge will present a uniformly sharp edge to the block or sharpen

Re-trim the block

Readjust knife and block

Repeat impregnation using pure wax

Sections are compressed wrinkled or jammed

Re-sharpen the knife

Cool the block on ice water

until firm

Clean the knife edge

Re-adjust thickness of section

Tighten screw

Reduce the tilt

Decrease cutting speed for

every thin section

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

Trim the block edges until they are parallel

Sections are squashed (width of each section is less than that of block

Re-embed in freshly filtered wax if necessary

Once embedded in paraffin wax, decalcification is impractical; use a base sledge microtome with a wedge knife

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

A hole is formed in the section

Re-embed in freshly filtered wax if necessary

Once embedded in paraffin wax, decalcification is impractical; use a base sledge microtome with a wedge knife

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

Sections of unequal thickness are produced

Reduce the tilt

Tighten the screw

Cut blocks into smaller

fragments

Soften the blocks in detergent or phenol

Sections adhere to the knife or other parts of the microtome

Breathe out or blow gently on the block and knife to breakup static electricity , or boil water in the room to increase humidity

Clean knife edge

Sharpen the knife

Reduce tilt

Ribbon is split or lengthwise vertical scratches are seen on sections

Sharpen the knife

Re-embed in filtered wax

Clean knife edge with xylene

Reduce the tilt

Sections are lifted from the knife on the upstrokes

Reduce the tilt

Sharpen the knife

Cool paraffin wax in ic

Resistance is felt on the lower part of the section during cutting

Increase the tilt

Horizontal or parallel lines or furrows across the section (chatters) are seen, forming thin and thick zones

Treat with phenol during processing or collodionize

Reduce tilt

Restore moisture of tissue by

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

Resharpen the knife/blade

Decrease cutting speed

Section cut is sometimes thin sometimes thick

Sharpen the knife

Adjust the knife

Reduce tilt

Tighten adjusting and locking

screw

Increase the tilt

Knife makes a hard metallic scrapping or ringing sound on backstroke, when section is cut

Readjust the angulation of the knife

Take fresh block treated with phenol during processing

Change the knife

Frozen tissue crumbles and comes off the block holder when cut

Refreeze the tissue block

Frozen tissue chips into fragments when cut

Warm the tissue with the fingers

Pleated, folded and crooked ribbons

Trim the block parallel to the other edge or re-embed them in molds so that the edge of blocks is parallel to the knife edge

Try another part of the knife edge

Brittle or Hard tissues

Tissue may be softened by soaking in a small dish or bowl containing water with detergent, phenol or molliflex

Clearing agent turns milky as soon as tissue is placed in it

Repeat dehydration with absolute alcohol then clear again

On trimming, tissue smells of clearing agent

Block is trimmed down nearest to the tissue. Remaining wax is melted on embedding oven and paraffin impregnation is repeated, changing the paraffin at least once before blocking

Tissue is opaque, section cutting is difficult due to presence of alcohol

Repeat clearing; if object has already been embedded, prolong clearing up to 12 hours, then re-embed

Tissue shrinks away from wax when trimmed

Repeat the whole procedure

Tissue is soft when block is trimmed

Repeat the whole procedure

Airholes are found on tissue during trimming

Repeat Impregnation

On trimming wax appears crystalline

Re-embed in freshly filtered wax

Paraffin block, after cooling is moist and crumbles

Repeat paraffin impregnation, then re-embed

Inadequate processing of fatty tissues

Fatty tissues should be sampled and cut thinly for easy reagent action. Ethanol is a poor fat solvent. To ensure complete dehydration, a far better lipid solvent (acetone or isopropanol) should be inserted before using the final absolute ethanol

Hard, dense tissues

Treat fixed tissues using 4% aqueous phenol for 24-72 hrs to soften

On embedding, tissue is soft and mushy in the center

At gross, cut thin sections especially fatty tissues, ensure complete fixation