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Histology Spring 2020 > Processing and Decal > Flashcards

Processing and Decal Flashcards

1
Q

What is tissue processing?

A

Takes fixed tissue; dehydrates, clears, and infiltrates so it can be embedded in paraffin for sectioning

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2
Q

Why do we process tissue?

A

To give it support for sectioning

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3
Q

Dehydrant

A

Removes water from the fixed tissue

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4
Q

Clearing agent

A

Removes the dehydrant (alcohol) and makes the tissue receptive to paraffin

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5
Q

Infiltrating medium

A

Typically paraffin, infiltrates the tissue prior to embedding

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6
Q

What is the difference between an open or closed tissue processing system?

A

Open allows for the evaporation of potentially hazardous chemicals, like xylene

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7
Q

Properties and Actions of Ethanol as a dehydrant

A

Clear, colorless, flammable
fast, reliable, best dehydrant
hydrophilic: mixes well with water

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8
Q

Properties and Actions of Methanol as a dehydrant

A

Clear, colorless, flammable, poisonous
Rarely used alone, similar to ethanol
preferred for blood smears

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9
Q

Properties and Actions of Isopropyl Alcohol as a dehydrant

A

Toxic, flammable

Doesn’t harden or shrink tissue as much as Ethanol

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10
Q

Properties and Actions of Acetone as a dehydrant

A

Rapid, less expensive
Excessive shrinkage
Absorbs water when exposed to air
volatile

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11
Q

Is Ethanol advantageous for tissue processing?

A

Ethanol is the best for dehydration during processing

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12
Q

Is Methanol advantageous for tissue processing?

A

Primarily used for fixing blood smears

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13
Q

Is Isopropyl Alcohol advantageous for tissue processing?

A

Excellent substitute for Ethanol, however it can’t be used to make eosin stains

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14
Q

Is Acetone advantageous for tissue processing?

A

Yes for speed, no for open processors due to ease of evaporation

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15
Q

Properties and Actions of Xylene as a clearing Agent

A 
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
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16
Q

Properties and Actions of Toluene as a clearing Agent

A

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

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17
Q

Properties and Actions of Benzene as a clearing Agent

A

Very fast, doesn’t over-harden as much as Xylene
Hardens muscle, tendon, and uterus more than toluene
Very volatile, very toxic, carcinogenic

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18
Q

Properties and Actions of Chloroform as a clearing Agent

A

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

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19
Q

Properties and Actions of Cedarwood oil as a clearing Agent

A

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

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20
Q

Properties and Actions of Dioxane as a clearing Agent

A

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

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21
Q

Properties and Actions of Limonene as a clearing Agent

A 
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
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22
Q

Is Xylene advantageous for tissue processing?

A

Yes, due to speed and hydrophobic nature

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23
Q

Is Toluene advantageous for tissue processing?

A

Doesn’t overharden as much as xylene

More likely to have water contamination

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24
Q

Is Benzene advantageous for tissue processing?

A

No; although very fast acting it is too toxic, volatile, and carcinogenic to be used in a routine histology lab

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25
Is Chloroform advantageous for tissue processing?
No; slow, easily absorbs water from air, ad carcinogenic | Good for muscle, tendon, and uterus
26
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
27
Is Dioxane advantageous for tissue processing?
No, strong odor, can distort delicate tissues, flammable and a suspected carcinogen
28
Is Limonene advantageous for tissue processing?
Yes, it hardens tissues less than xylene and is hydrophobic, however it contaminates paraffin more than Xylene
29
Miscible
two substances that are soluble in all proportions, they should form a clear solution
30
What are the three universal solvents?
Reagents that both dehydrate and clear | Dioxane, tetrahydrofuran, tertiary butanol
31
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
32
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
33
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
34
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
35
Importance of using graded alcohols in tissue processing
70-95-100% alcohol gradation reduces tissue shrinkage during dehydration
36
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
37
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
38
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
39
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
40
When and how should decal occur?
Should be done after fixation but before processing | Routine histology labs typically use an acid for decal
41
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
42
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
43
Function of acid decalcifiers
Calcium salts dissolve, then ionize, then migrate into the surrounding solution
44
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
45
How does heat effect decal?
heat speeds up the decal process
46
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
47
Methods of determining decal endpoint
Mechanical: bend, scratch Chemical: test for calcium ions in solution Radiographic: x-ray to visually confirm decal
48
Why is it important to remove decal reagents before processing the tissue?
Because the acid can affect pH of the processor
49
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
50
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
51
Chelating agents
organic compounds that can bind certain metals ex: EDTA binds only ionized calcium Very slow, but good for enzyme reactions on sections
52
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
53
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 ```
54
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
55
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 ```
56
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
57
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
58
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)
59
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
60
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
61
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
62
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
63
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
64
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

Histology Spring 2020 (14 decks)

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