Frozens and Cytology EX 1

Most tissue temp

-13 to -20

Fatty and softer tissue (breast, skin, pancreas) temp

-25 to -30

Dryer/harder tissue (brain, liver, spleen, lymph node, bloody tissues) or fixed tissue temp.

-5 to -15

Peltier element

-60 C

Rapid freezing

If anti-roll plate angle is below…

sections curls on edge of knife

If anti-roll plate angle is above…

the tissue hits the plate on downstroke

Freeze artifact

Caused by slow freezing

Ice crystals become holes in tissue

Common in brain, lung, kidney, bowel

Liquid Nitrogen artifact

Liquid nitrogen vapor bubbles

Best method for freezing

Isopentane chilled in liquid N

Slowest freezing method

Cryostat freezing and causes most freeze artifacts

Freeze spray

Freezes tissue but easily produces freeze artifact.

Will also aerosolize infectious tissue

Frozen storage

Short term - in cryostat or regular freezer

Extended - store in -80C freezer.

Wrap in foil

Difficulty getting a section

Alter temp

Sharpen/change blade

Rotate spec (shortest path is usually best)

Tighten/oil/clean components

Section shreds causes

Dull knife

Wrong temp

Tissue has hard components (calcifications, etc.)

Compression

Blade too warm

Blade holder or knife surface dirty

Incorrect knife angle

Dull blade

Thick/Thin section causes

Loose blade/specimen chuck

Dull Blade

Wrong temp

Incomplete section causes

Insufficient facing

Tissue depletion

Improper orientation

Varying tissue densities (different optimal temps)

Tissue chips out causes

Aggressive cutting

Hard tissue

Loose cryostat

Tissue or OCT is too cold (especially when freezing in isopentane)

Sections not laying flat causes

Anti-roll plate is not positioned correctly/dirty/marred

Frost on blade

Static

Mohs advantages

Preserves tissue, minimizes re-growth of tumor

Less scarring

Reliable and accurate

Mohs disadvantages

Time consuming

Costly

Diagnostic cytology

Assessment of morphological details and chromatin patterns

Determines if cells are normal/abnormal

Nucleus:Cytoplasm ratio

Liquid based cytology

GYN spec received in preservative or fixative

Cell block

Aggregate cells processed histologically

And/or when IHC needed

GYN Spec

Female genital spec.

PAP smears (slide smears and liquid brushings)

Non-GYN specs

Body fluids, discharges, CNS fluid, washings/brushings, urine, FNAs, Cyst aspirates

Fixation of choice in Cytology

95% ETOH

Will dehydrate and sharpen nuclear membrane.

Methanol as a fixative

100% is used as alt to 95% ETOH

80% isopropanol as a fixative

Shrinks cells more than 95% ETOH

Cytolyt as a fixative

Methanol based, less shrinkage than ETOH

Commercial spray fixatives

Alcohol - fix

Acetone - dry

Polyethylene - protective coating (remove w/alcohol before staining)

Saccomanno Fluid as a pre-fixative for transportation

50% ETOH + Carbowax

Alcoholic saline as a pre-fixative for transportation

Equal Saline + 50% ETOH

Commercial transport fluid as a pre-fixative for transportation

Alcoholic based

Pull apart method - Prep smear

Preferred, nice monolayer, cells will not dry out

Cross Hatch - Preparing smear

Use loop to smear specimen on slide

Crush - Preparing smear

Used for mucoid spec

For sparsely cellular specimens…

Centrifuge to separate cells (button) and supernatant

For bloody specimens…

Lyse RBCs w/commercial lysing agent or with Carnoy’s acetic alcohol solution

For Cell adhesion to slide…

Spray adhesives, but not needed with pull-apart method

To remove excess mucoid…

Use a mucolytic agent.

DTT (Dithiotheritol) → reduces protein disulfide bonds

Cell Block Preparation steps

Centrifuge → Pour off supernatant → Fix button in 95% ethanol then 10% NBF → Embed in histogel/agar or infiltrate and embed w/paraffin

Why use cell blocks?

Diagnostic information - correlation w/smears

Special stains and IHC - Cell block spec can withstand harsh chemical contact better than smears

PAP Stain Reagents

Hematoxylin - nuclear

OG (Orange G) - Keratin, squam cells

EA (Light Green and Eosin) - Cytoplasmic components

Hematoxylin as a PAP Stain Reagent

Nuclear detail

OG (Orange G) as a PAP Stain Reagent

Keratin, squam cells

EA (Light Green and Eosin) as a PAP Stain Reagent

Cytoplasmic components

PAP Stain purpose

Visualize and differentiate cell components.

Provides nuclear and cytoplasmic morphology

More info about cell blocks…

A cell block is a method of preparing cytology material so that it can be processed, sectioned, stained and viewed as a histology section. 

It can provide diagnostic information in addition to that obtained from cytology smears. 

Also, it is easier to do special stains if needed, including IHC, on a cell block than on additional smears, because smears often require adaptations of the staining protocols and different controls. 

More on cell block prep

A cell block is prepared with material remaining after the cytology smears have been made. 

The quality or quantity of the smears should never be sacrificed to make a cell block. 

The method chosen is dependent upon the characteristics of the specimen. 

If a cytology specimen contains small fragments of tissue, the fragments should be carefully picked out and submitted as a cell block. 

Clots or heavy mucus in specimens should be used to prepare cell blocks.  Loose cells are difficult to contain, embed, and section and therefore make poor cell blocks. 

A method should be used with all loose cellular material to “block” it together so that it can be handled “en masse”.