Processing and Decal Flashcards
What is tissue processing?
Takes fixed tissue; dehydrates, clears, and infiltrates so it can be embedded in paraffin for sectioning
Why do we process tissue?
To give it support for sectioning
Dehydrant
Removes water from the fixed tissue
Clearing agent
Removes the dehydrant (alcohol) and makes the tissue receptive to paraffin
Infiltrating medium
Typically paraffin, infiltrates the tissue prior to embedding
What is the difference between an open or closed tissue processing system?
Open allows for the evaporation of potentially hazardous chemicals, like xylene
Properties and Actions of Ethanol as a dehydrant
Clear, colorless, flammable
fast, reliable, best dehydrant
hydrophilic: mixes well with water
Properties and Actions of Methanol as a dehydrant
Clear, colorless, flammable, poisonous
Rarely used alone, similar to ethanol
preferred for blood smears
Properties and Actions of Isopropyl Alcohol as a dehydrant
Toxic, flammable
Doesn’t harden or shrink tissue as much as Ethanol
Properties and Actions of Acetone as a dehydrant
Rapid, less expensive
Excessive shrinkage
Absorbs water when exposed to air
volatile
Is Ethanol advantageous for tissue processing?
Ethanol is the best for dehydration during processing
Is Methanol advantageous for tissue processing?
Primarily used for fixing blood smears
Is Isopropyl Alcohol advantageous for tissue processing?
Excellent substitute for Ethanol, however it can’t be used to make eosin stains
Is Acetone advantageous for tissue processing?
Yes for speed, no for open processors due to ease of evaporation
Properties and Actions of Xylene as a clearing Agent
Most common clearing agent Tends to overharden tissue after prolonged exposure Works rapidly Intolerant of water Flammable, hazardous (no sink disposal) Cloudy Xylene should be replaced
Properties and Actions of Toluene as a clearing Agent
Doesn’t overharden tissue as much as Xylene
Tissue can be submerged overnight without damage
Higher chance of atmospheric water contamination
Flammable, more volatile thank Xylene
Properties and Actions of Benzene as a clearing Agent
Very fast, doesn’t over-harden as much as Xylene
Hardens muscle, tendon, and uterus more than toluene
Very volatile, very toxic, carcinogenic
Properties and Actions of Chloroform as a clearing Agent
Makes tissue less brittle than Xylene
Penetrates slowly so clearing takes longer
Easily absorbs atmospheric water, must be contained
Better than all others for muscle, tendon, and uterus
Volatile, doesn’t make tissue transparent
Carcinogen
Properties and Actions of Cedarwood oil as a clearing Agent
Expensive and only used for special projects
Volatile with strong odor
Clears quickly after dehydration with 95% alcohol
Can stay in oil indefinitely
Hardens and damages tissue less than any other agent
Properties and Actions of Dioxane as a clearing Agent
Is a universal solvent; it can both dehydrate and clear
Bad for delicate tissue because strong currents during immersion can distort the tissue
toxic, strong odor, must have ventilation
flammable and suspected carcinogen
Properties and Actions of Limonene as a clearing Agent
Xylene substitute Hardens tissues less than Xylene Contaminates paraffin more than Xylene Irritant when concentrated Can't go down drain because it is immiscible with water
Is Xylene advantageous for tissue processing?
Yes, due to speed and hydrophobic nature
Is Toluene advantageous for tissue processing?
Doesn’t overharden as much as xylene
More likely to have water contamination
Is Benzene advantageous for tissue processing?
No; although very fast acting it is too toxic, volatile, and carcinogenic to be used in a routine histology lab
Is Chloroform advantageous for tissue processing?
No; slow, easily absorbs water from air, ad carcinogenic
Good for muscle, tendon, and uterus
Is Cedarwood oil advantageous for tissue processing?
Yes, it hardens and damages tissue the least of any reagent, but it is prohibitively expensive and therefore only used for special experiments
Is Dioxane advantageous for tissue processing?
No, strong odor, can distort delicate tissues, flammable and a suspected carcinogen
Is Limonene advantageous for tissue processing?
Yes, it hardens tissues less than xylene and is hydrophobic, however it contaminates paraffin more than Xylene
Miscible
two substances that are soluble in all proportions, they should form a clear solution
What are the three universal solvents?
Reagents that both dehydrate and clear
Dioxane, tetrahydrofuran, tertiary butanol
Why is paraffin used in routine histology labs?
Because a large number of tissue blocks can be processed in a relatively short amount of time, serial sections (ribbon) are easily obtained, and because it is easy to perform routine and special stains
What happens to processing and H&E when water is present in the dehydration steps?
Water left over after dehydration disrupts the clearing process, which in turn disrupts infiltration, which can result in mushy tissue.
This can also result in poor or absent nuclear staining in H&E
How does pH of fixative affect the tissue processing unit?
pH of Zinc buffered formalin above 7 can cause precipitate in the processor chamber and tubing
special precautions for handling tissue processing reagents
Use the reagents in a closed processor, esp Xylene because it is flammable and the fumes are dangerous
Importance of using graded alcohols in tissue processing
70-95-100% alcohol gradation reduces tissue shrinkage during dehydration
what varies in a processing schedule based on type and size of tissue being processed?
The amount of time in the processor
larger tissues take longer
kidney or liver core 3hrs
biopsy 6hrs
standard surgical specimen 13hrs
high fat/lipid like breast and brain 22hrs
some of these can be sped up using a microwave, esp biopsies
what varies in a processing schedule based on type and size of tissue being processed?
Small biopsies take a short program, while large, bloody, or fatty tissues take a long 8-15 hour program
What happens when dehydration or clearing is inadequate?
Clearing agent can’t fully penetrate due to presence water, which in turn displaces paraffin, resulting in mushy or soft tissue
What happens when dehydration or clearing is excessive?
May result in hard or brittle tissue that is difficult to section
Also causes microchatter around the edges of tissue on H&E sections, common for biopsies
When and how should decal occur?
Should be done after fixation but before processing
Routine histology labs typically use an acid for decal
Why does decal need to be carefully monitored?
Because it can be easy to over decalcify a tissue, which reduces it’s quality and stainability
Pros and cons of acid decal
Decal makes hard tissues like bone easier to cut so you don’t damage your microtome blades
But over decal can damage tissue and affect stain quality, esp of nuclei
Function of acid decalcifiers
Calcium salts dissolve, then ionize, then migrate into the surrounding solution
List of acid decalcifiers and their advantages
HCl: rapid, requires thorough washing before decal and careful monitoring
Nitric Acid: rapid, requires careful monitoring and can deteriorate stainability
Formic acid: slower but yields better staining
How does heat effect decal?
heat speeds up the decal process
Ion exchange resins
Formic acid over an ammoniated salt of a sulfonated resin
ammonium ions are exchanged for calcium ions which keeps the solution free of calcium and speeds up the process, which results in excellent staining
Methods of determining decal endpoint
Mechanical: bend, scratch
Chemical: test for calcium ions in solution
Radiographic: x-ray to visually confirm decal
Why is it important to remove decal reagents before processing the tissue?
Because the acid can affect pH of the processor
How to decal a block with localized calcification?
Once the block as been faced, soak it in 1% HCl for 30-60 minutes, rinse in ice water, blot dry, and section
Electrolyte decal
formic acid and HCl paired with an electroporating device
bone is attached to the anode (-), current is passed through, and calcium ions(+) are attracted to the cathode (-)
High potential for tissue destruction and loss of stainablility
Chelating agents
organic compounds that can bind certain metals
ex: EDTA binds only ionized calcium
Very slow, but good for enzyme reactions on sections
Case Study: What went wrong?
Breast case, tissue disruption of loose connective tissue in all sections of the case, not a fixation problem because other tissues in the same processing batch are acceptable
Was the breast processed on the proper fatty program with other fatty tissue?
Possibly grossed too thickly resulting in mushy tissue
Case Study: Rule out 5 potential causes
Breast case, tissue disruption of loose connective tissue in all sections of the case, not a fixation problem because other tissues in the same processing batch are acceptable
Because other tissues in batch look ok: Improper temperature Contaminated reagents Diluted reagents Equipment failure Correct order of reagents on processor
Case Study: How to fix?
Trimming a bone block, not completely decalcified
First try to surface decalcify and reattempt to section
If this fails, melt down the block, un-process, then decalcify again, reprocess, re-embed, and attempt to section
Case Study: What issues will incompletely decalcified bone cause while sectioning?
Chunking of specimen Difficult to cut Shredding Blade damage Knife marks Difficult to get sections
Case Study: What went wrong?
Bone H&E has loss of nuclear staining and detail despite being decalcified according to protocol
Over decalcified or the acid used was too strong
Case Study: How to avoid compromising future bone sections?
Carefully monitor decal to prevent over or under
Better to do in short 30 minute stages and test section-ability
Use thorough endpoint detection
Troubleshooting:
While embedding you notice most of the tissue is mushy and under-processed, what is the FIRST thing you check?
Check to see if the wrong processing program was used
Ex: processing breast (fatty/long) on a biopsy schedule (short)
What are two causes of tissue that looks greasy and explodes when the ribbon is placed on the water bath?
Water left over from incomplete dehydration causes insufficient clearing and infiltration so that xylene is still present in the tissue
under-processed (time)
too thick at grossing
Why should the alcohols on the tissue processor be changed on a regular basis?
Because they will gradually become contaminated from water in the tissues being processed
We want to avoid dilution and contamination of the dehydrating alcohols
Why is tissue dehydration necessary?
We must remove water to make tissue permeable to clearing by xylene and by extent infiltrate-able by paraffin for embedding
What is a disadvantage of using heat during every step of processing?
Heat can cause evaporation of alcohols and potentially denature tissue
Only heat at the paraffin infiltration stage, otherwise you will get hard, brittle tissue that is difficult to section
What is the advantage of dehydrating tissues in graded alcohols of increasing concentration instead of going directly into absolute alcohol?
This reduces tissue shrinkage typically caused by absolute alcohol
Why is it important to maintain adequate volumes of reagents in the tissue processor?
If the cassettes aren’t fully submerged in the tissue processor, the tissue can dry out, or some reagents may not be completely removed or rinsed away between steps
