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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
Q

Is Chloroform advantageous for tissue processing?

A

No; slow, easily absorbs water from air, ad carcinogenic

Good for muscle, tendon, and uterus

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26
Q

Is Cedarwood oil advantageous for tissue processing?

A

Yes, it hardens and damages tissue the least of any reagent, but it is prohibitively expensive and therefore only used for special experiments

27
Q

Is Dioxane advantageous for tissue processing?

A

No, strong odor, can distort delicate tissues, flammable and a suspected carcinogen

28
Q

Is Limonene advantageous for tissue processing?

A

Yes, it hardens tissues less than xylene and is hydrophobic, however it contaminates paraffin more than Xylene

29
Q

Miscible

A

two substances that are soluble in all proportions, they should form a clear solution

30
Q

What are the three universal solvents?

A

Reagents that both dehydrate and clear

Dioxane, tetrahydrofuran, tertiary butanol

31
Q

Why is paraffin used in routine histology labs?

A

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
Q

What happens to processing and H&E when water is present in the dehydration steps?

A

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
Q

How does pH of fixative affect the tissue processing unit?

A

pH of Zinc buffered formalin above 7 can cause precipitate in the processor chamber and tubing

34
Q

special precautions for handling tissue processing reagents

A

Use the reagents in a closed processor, esp Xylene because it is flammable and the fumes are dangerous

35
Q

Importance of using graded alcohols in tissue processing

A

70-95-100% alcohol gradation reduces tissue shrinkage during dehydration

36
Q

what varies in a processing schedule based on type and size of tissue being processed?

A

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
Q

what varies in a processing schedule based on type and size of tissue being processed?

A

Small biopsies take a short program, while large, bloody, or fatty tissues take a long 8-15 hour program

38
Q

What happens when dehydration or clearing is inadequate?

A

Clearing agent can’t fully penetrate due to presence water, which in turn displaces paraffin, resulting in mushy or soft tissue

39
Q

What happens when dehydration or clearing is excessive?

A

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
Q

When and how should decal occur?

A

Should be done after fixation but before processing

Routine histology labs typically use an acid for decal

41
Q

Why does decal need to be carefully monitored?

A

Because it can be easy to over decalcify a tissue, which reduces it’s quality and stainability

42
Q

Pros and cons of acid decal

A

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
Q

Function of acid decalcifiers

A

Calcium salts dissolve, then ionize, then migrate into the surrounding solution

44
Q

List of acid decalcifiers and their advantages

A

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
Q

How does heat effect decal?

A

heat speeds up the decal process

46
Q

Ion exchange resins

A

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
Q

Methods of determining decal endpoint

A

Mechanical: bend, scratch
Chemical: test for calcium ions in solution
Radiographic: x-ray to visually confirm decal

48
Q

Why is it important to remove decal reagents before processing the tissue?

A

Because the acid can affect pH of the processor

49
Q

How to decal a block with localized calcification?

A

Once the block as been faced, soak it in 1% HCl for 30-60 minutes, rinse in ice water, blot dry, and section

50
Q

Electrolyte decal

A

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
Q

Chelating agents

A

organic compounds that can bind certain metals
ex: EDTA binds only ionized calcium
Very slow, but good for enzyme reactions on sections

52
Q

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

A

Was the breast processed on the proper fatty program with other fatty tissue?

Possibly grossed too thickly resulting in mushy tissue

53
Q

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

A 
Because other tissues in batch look ok:
Improper temperature 
Contaminated reagents 
Diluted reagents
Equipment failure 
Correct order of reagents on processor
54
Q

Case Study: How to fix?

Trimming a bone block, not completely decalcified

A

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
Q

Case Study: What issues will incompletely decalcified bone cause while sectioning?

A 
Chunking of specimen
Difficult to cut
Shredding 
Blade damage
Knife marks
Difficult to get sections
56
Q

Case Study: What went wrong?

Bone H&E has loss of nuclear staining and detail despite being decalcified according to protocol

A

Over decalcified or the acid used was too strong

57
Q

Case Study: How to avoid compromising future bone sections?

A

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
Q

Troubleshooting:

While embedding you notice most of the tissue is mushy and under-processed, what is the FIRST thing you check?

A

Check to see if the wrong processing program was used

Ex: processing breast (fatty/long) on a biopsy schedule (short)

59
Q

What are two causes of tissue that looks greasy and explodes when the ribbon is placed on the water bath?

A

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
Q

Why should the alcohols on the tissue processor be changed on a regular basis?

A

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
Q

Why is tissue dehydration necessary?

A

We must remove water to make tissue permeable to clearing by xylene and by extent infiltrate-able by paraffin for embedding

62
Q

What is a disadvantage of using heat during every step of processing?

A

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
Q

What is the advantage of dehydrating tissues in graded alcohols of increasing concentration instead of going directly into absolute alcohol?

A

This reduces tissue shrinkage typically caused by absolute alcohol

64
Q

Why is it important to maintain adequate volumes of reagents in the tissue processor?

A

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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