Grossing, Processing, and Instrumentation Flashcards
(122 cards)
1
Q
Could limonene be substituted for xylene as a clearing reagent?
A
Yes. it is a xylene substitute.
2
Q
Why is limonene not used more than xylene as a clearing reagent in laboratories.
A
You have to change the paraffin more often due to contamination.
Also, it is an irritant and sensitizer.
It can cause headaches, difficulty breathing, and allergic skin reactions.
3
Q
Define dehydration in terms of tissue processing.
A
The removal of free water from tissue in order to embed in a non-aqueous medium. This is usually done with graded alcohols.
4
Q
What is clearing in tissue processing?
A
The removal of the dehydrating reagent (usually alcohol) in order to replace it with the infiltration medium (usually paraffin).
5
Q
Why must we infiltrate tissue with the embedding medium prior to embedding?
A
The embedding medium must permeate the tissue in order to keep it in place in the block during microtomy.
6
Q
True or False: Tissue can remain in hot paraffin indefinitely.
A
False: Tissue should remain in hot paraffin for the MINIMUM amount of time necessary for infiltration. This is to prevent heat damage.
7
Q
What is the correct temperature of molten paraffin in the tissue processor?
A
2° to 4° above the melting point of the paraffin.
8
Q
What is the optimal temperature range of the microtomy waterbath.
A
5° to 10° below the melting point of the paraffin.
9
Q
What is the most popular medium for tissue embedding?
A
Paraffin
10
Q
What is embedding?
A
Encasing tissue into a block that can be cut thinly on a microtome.
11
Q
What do histotechs mean by universal solvent?
A
In histochemistry, a universal solvent is one that can replace 2 solutions. Thus reducing the number of processing steps. This is too jarring for delicate tissue, though.
12
Q
What happens if formalin salts get into patient tissue?
A
Shredding during microtomy.
13
Q
What do histotechs mean by formalin salts?
A
The buffering salts used in 10% NBF. (Usually sodium phosphate monobasic and sodium phosphate dibasic)
14
Q
Why is the warm water flush done on the first 3 to 5 stations of the tissue processor.
A
The buffers used in 10% NBF are soluble in water but precipitate out in alcohol. Flushing prevents these salts from accumulating in the tubes.
15
Q
Can formalin salts precipitate onto the tissue as well?
A
Yes, if the first alcohol after formalin is 95% or higher, the phosphate buffers can precipitate onto and into the tissue.
16
Q
Name 3 dehydrating agents.
A
All alcohols, Acetone, Tetrahydrofuran, Dioxane.
17
Q
Which is the faster dehydrant, Alcohol or Acetone?
A
Acetone. It not only removes water faster but is itself replaced quickly by clearing agents. However, for this reason, it is a higher risk for overprocessing.
18
Q
Name 3 clearing agents.
A
Xylene, Toluene, Tetrahydrofuran, Dioxane, Limonene, Benzene, Chloroform, Cedarwood Oil, Clove Oil, and Aliphatic Hydrocarbons.
19
Q
Name 3 universal solvents.
A
Acetone, Dioxane, Tertiary Butanol, and Tetrahydrofuran (or THF).
20
Q
How is the term cross-section used when embedding?
A
A transverse section. In vessels, it will demonstrate the lumen. This is opposed to a tangential section that would only graze the surface of the specimen.
21
Q
When would a grosser submit tissue for decalcification?
A
After fixation in order to remove calcium for paraffin embedding.
22
Q
Why is FFPE (formalin-fixed, paraffin embedded) tissue submitted for decalcification after fixation?
A
The tissue will continue to deteriorate during decalcification.
23
Q
If tissue for paraffin embedding is submitted for decal after fixation, when is tissue meant for embedding in plastic decaled?
A
It is unnecessary to decal tissue that will be embedded in plastic as the hardened plastic can support undecalcified bone. In fact, tissue with suspected metabolic bone disease is submitted undecalcified in plastic for this reason.
24
Q
Name 3 methods of decalcification.
A
Acid, Ion-exchange resins, electrolysis.
25
What is the primary microscope used in the histopathology laboratory?
The light microscope.
26
A compound microscope contains 2 magnifying lenses. What are they called?
Objective and ocular.
27
Name some of the objective lenses and give their magnifications.
Scanning (2.5X to 4X), intermediate (10X to 20X), high-powered dry lens (40X to 45X), and oil immersion lens (90X to 100X).
28
What is resolving power?
A measurement of the least distance between 2 objects that allows them to be SEEN AS 2 objects.
29
What is the resolving power of the light microscope?
0.2 microns.
30
How much are ocular lenses magnified?
5X to 15X.
31
How do you compute the magnification of a particular slide under a compound microscope?
By multiplying the ocular lens magnification by the objective lens magnification.
32
Agitation can be used on open and closed processors as well as manual processing. Why would we want it?
It facilitates fluid exchange.
33
When 10%NBF is used, why should dehydration begin with an alcohol less than 70% concentration?
Because when fixing with zinc formalin or 10% NBF, precipitate can form in the processor tubing and/or patient tissue.
34
How should bone be embedded?
Diagonally so that the microtome blade contacts only a small surface area of the tissue at a time.
35
Describe 2 applications for a microwave oven in the histology laboratory.
Fixation, processing, and staining.
36
How are tubes embedded?
On end.
37
What is the best way to embed tissue with layers?
On edge.
38
Name two types of electron microscopes.
Scanning and transmission.
39
What are the three types of solidification?
Polymerization (paraffin and carbowax), Evaporation (celloidin), and Crystallization (resins and plastics).
40
What is freeze artifact?
Holes in the tissue where ice crystals formed.
41
Isopropanol is often used in microwave processing. Its use isn't restricted by the government, and it doesn't shrink tissue as much as ethanol. So why isn't it our go-to alcohol in the histo lab?
Eosin is insoluble in isopropyl alcohol. And Eosin IS 1/2 of our go-to stain.
42
What is birefringence?
Double refraction. It happens when a crystal splits a single ray of light into 2 rays with differing refractions. It can be observed using a polarizing microscope.
43
What is plane of section?
The position of the microtome cut in relation to the anatomical structures in the tissue.
44
What is tissue processing?
The removal of free water from tissue through a series of graded alcohols in order to replace it by an infiltrating media.
45
Why do we need to "fix" and"process" the tissue before staining it?
The goal of both tissue fixation and processing is to make the tissue firm enough for thin sectioning without disturbing the morphology.
46
What is a microscope with only one lens called?
A simple microscope rather than a compound microscope that has two lenses. Aka a magnifying glass.
47
After fixation with NBF, what concentration should the 1st alcohol be?
No more than 65%.
48
How is a polarizing microscope used in the histology laboratory?
To view urate crystals, amyloid stained with Congo Red, and fixation pigments. Urate crystals are shown.
49
What is a hydrometer?
An instrument used to measure the water content in alcohol.
50
What is micrometry?
Microscopic measurement using an eyepiece and a stage micrometer.
51
What is the difference between bright field and dark field microscopy?
Bright field shows images on a light background, and dark field shows images on a dark background.
52
True or False: An ultrasection is a paraffin section with no wrinkles.
False. An ultrasection is a very thin tissue section cut on an ultramicrotome for EM.
53
What is the preferred size and shape of a block cut on an ultramicrotome?
Trapezoid with the longest side being no more than 1 mm.
54
True or False: You can write on both cassettes and slides with a pencil, and it won't come off during processing or staining.
True
55
Name 3 factors that affect processing speed.
Agitation, Heat, Vacuum, Viscosity of Reagents.
56
When decalcifying using acids, what is the ratio of reagent to tissue?
20:1, the same as it is for fixation.
57
True or False: All acid decalcifiers affect tissue staining.
True. pH is very important in staining, and all acids affect staining to some degree.
58
Is it possible to decalcify with weaker acids?
Yes, weak acids such as formic acid take longer to decalcify, but even picric acid and acetic acid can decalcify microcalcifications. This is why histotechs must be careful choosing fixatives with acids when trying to demonstrate microcalcs. (As with breast needle cores)
59
Using a simple acid decalcifying method, calcium ions are released from bone, then ionize with the acid to produce calcium salt. Why doesn't the solution become saturated with calcium salt?
It does. That's why you must change the decalcifier often.
60
Do agitation, vacuum, and heat help with decalcifying as they do with fixation and processing?
Agitation and vacuum are great, but heat is a no-no when decalcifying. Actually, heat should be used cautiously in fixation and processing.
61
What is considered the best method for decalcifying bone?
Ion exchange resin. It's faster, the solution doesn't need to be changed often, and you don't have to worry about leaving the tissue in the reagent too long.
62
How does ion exchange work on decalcification?
You place a sulfonated resin of ammoniated salt in the vessel and cover it with formic acid. The ammonium ions are exchanged with the calcium ions exiting the bone, and the solution itself doesn't become saturated.
63
The electrolytic method of decalcification works within a few hours. Why don't most labs decalcify with this method?
The heat pretty much destroys the tissue.
64
What's the decalcifying agent that uses chelation?
EDTA (or Ethylenediaminetetracacetic acid) is an organic compound that directly binds with calcium ions. This is called Chelation. It is much better for tissue morphology, and it can be combined with other acids and fixatives for a good effect. However, it is very slow.
65
What is meant when we say tissue was underprocessed?
Enough of the free water was not removed, causing incomplete clearing and infiltration.
66
What is meant by overprocessing tissue?
Biologically bound water was removed during processing, causing microchatter.
67
What is the magic percentage of alcohol for tissue storage?
70%. This is isotonic for tissue storage. Lower and the tissue will swell. Higher and the tissue will shrink.
68
Why is reagent alcohol not used in the EM lab?
Methanol content. Same reason EM labs don't fix with formalin.
69
Most histo labs add eosin to one of the last dehydrating alcohols. Is that a good idea?
Sure. Unless you are going to perform IF. (It could cause autofluorescence) Adding eosin this way makes the biopsies easier to see when embedding and cutting.
70
Is Anatech's Pro-Soft a good dehydrant for biopsies?
Yes, it is. It doesn't remove bound water and works well with the Peloris tissue processing method.
71
Why is clearing called clearing?
Clearing reagents have the same refractive index as tissue, so it looks translucent.
72
What step in tissue processing could cause damage to the tissue.
Every step in tissue processing, if not monitored correctly, could damage tissue. Too long in any step can cause tissue to overharden and become brittle. This is either due to extended time or the addition of heat at any step. Additionally, in paraffin processing, lipid extraction occurs during dehydrating and clearing.
73
Is Xylene an Aromatic Hydrocarbon?
Yes, so are Benzene and Toluene (think smelly). Not to be confused with Aliphatic Hydrocarbons, which are xylene substitutes.
74
Can chloroform be used as a clearing reagent?
Yep.
75
Can you store tissue in Essential Oils, such as Cedarwood Oil, Clove Oil, Sandalwood Oil, and Wintergreen Oil?
Yes. You would use the essential oil for clearing and just not infiltrate them until needed.
76
Name 3 infiltrating mediums.
Paraffin, Carbowax, Celloidin, Plastic
77
What do Beeswax, Rubber, Bayberry Wax, and Plastic have in common?
They are all paraffin additives.
78
What is Polyethylene Glycol?
Carbowax, a water soluble wax.
79
True or False: Celloidin hardens through crystallization.
False. It solidifies using evaporation.
80
How do plastics and resins solidify?
Crystallization.
81
What is the most common microtome for FFPE (formalin fixed paraffin embedded) tissue?
The rotary microtome.
82
What are the most obvious differences between a rotary microtome and a sliding microtome?
The rotary microtome moves the BLOCK vertically, and the sliding microtome moves the BLADE horizontally. Also, the sliding microtome is made for whole mounts and doesn't ribbon.
83
What type of knives are used in EM, and why?
Glass and Diamond. Because glass and diamond are very sharp and cut thinner sections.
84
What type of knife is used for plastic embedded tissue?
A Ralph (glass) knife.
85
What is the shape of a Ralph knife when it is being used to cut ultrathin sections?
A triangle.
86
Nowadays, knives have been replaced by disposable blades. Do they come in different sizes?
Yes, high and low profile.
87
On a rotary microtome, what is the correct clearance angle between the bevel of the blade and the block face?
2-5°
88
What can happen during microtomy when the clearance angle between the blade and the block is too slight? (Blade tilt is too verticle)
Skipped sections, thick and thin, wrinkles
89
What can happen during microtomy when the clearance angle between the block and the blade is too large? (Blade tilt too horizontal)
Chatter, microchatter, washboarding, trouble getting a ribbon
90
Why should the floatation, or hot water, bath be filled with hot water?
It helps keep it free of air bubbles.
91
True or False: The hot water bath surface needs to be cleaned after every block.
True.
92
True or False: Waterbaths should be cleaned once a week.
False. Floatation baths should be cleaned with hot water, soap, and bleach after every use. This is to prevent the growth of mold and/or bacteria.
93
True or False: Slides go in the oven before staining in order to melt the paraffin before deparaffinization.
False. Slides must be dried completely (and preferably vertically) so that the tissue is properly adhered before staining. This can be done overnight
or more quickly using an oven.
94
What happens when the slide drying oven temperature is too hot?
Darkened nuclei, nuclear bubbling, loss of nuclear detail.
95
What happens when the slide drying oven temperature is too low?
Tissue falls off slides because they were not completely dried.
96
How can you be sure the slide drying oven is at the right temperature?
Check the melting point of the paraffin. Should be less than 70°.
97
When should you choose to use charged slides (or slides with adhesives)?
When staining solutions are alkaline, for frozen sections, for IHC or IF, with decals, brains, or under processed tissue.
98
What are slide adhesives, and why do most labs no longer add adhesive to the waterbaths?
Gelatin, Poly-L-lysine, Silane, Albumin. They tend to cause background staining.
99
What are the three main mechanical causes of microtomy sectioning difficulties?
Dull blade, wrong clearance angle, something loose.
100
What can you do if you can't get a ribbon?
Blow on the block to warm it up or run a dowel stick over the blade to dull it a little.
101
Why might you see compression in your ribbons?
Dull blade, hard tissue, incomplete infiltration.
102
What should you do if your ribbons are compressed?
Get a new blade and make sure the block is cold.
103
What happens when the bottom off the block is not parallel to the blade?
The ribbon will curve or slant.
104
What happens when you rough your blocks too aggressively and don't "polish"?
Holes in the tissue. Fix by polishing the block.
105
When sectioning, why would you get a knife line?
A nick in the blade, debris in the paraffin, calcium, keratin, sutures, staples.
106
What is parched earth?
Cracks in tissue sections due to dryness.
107
What is the specific name for the person performing an autopsy or dissecting an organ.
The prosector. More commonly called the grosser. Sometimes called the cutter, although that term is also used for microtomists.
108
What are the most used anatomical planes?
Transverse (Superior/Inferior), Coronal (Anterior/Posterior), Sagittal (Medial/Lateral) are the 3 main anatomical directions. Other common directions are: Proximal/Distal, Superficial/Deep, Dorsal/Ventral and Caudal/Cephalad.
109
What is a skin excision?
A segment of skin that has been surgically removed. Usually circular or elliptical, but can be irregular.
110
What does it mean to breadloaf a specimen?
To slice from tip to tip.
111
Why do we breadloaf skin excisions?
So the pathologist can examine the layers of skin.
112
What is the margin of a skin excision?
The subcutaneous soft tissue layers and the area from the lesion to the edge of the excision.
113
What is a deep margin?
The margin located at the base of the specimen. In skin this would be the subcutaneous soft tissue.
114
Why are there clockface designations on cervical and skin excisions?
For orientation. The surgeon often places a suture in the specimen designating an analog clock position to correspond with an anatomical position.
115
Why are specimens inked?
To indicate orientation microscopically. In this way, the distance of a lesion to a margin can be measured and reported.
116
What is the difference between a positive and a negative margin?
Negative margins (also called clear) are when no cancer cells are close to the margins. Positive margins (also called involved) occur when cancer cells are close to or at the margin.
117
What words do pathologists used to descibe the function parts of an organ versus the supporting parts?
The functional part is called the parenchyma and any connective tissue, nerves or ducts are collectively called the stroma.
118
True or False: The only reason gluteraldehyde is not used for routine paraffin section, is because of possible subsequent staining difficulties such as PAS, IHC, or enzyme histochemistry.
False. Infiltration with paraffin is also difficult with gluteraldehyde. EM specimens are embedded in plastic.
119
What is the difference between a mastectomy and a lumpectomy?
A lumpectomy is a surgical procedure that removes a tumor and a surgical margin from the breast. A mastectomy removes the entire breast.
120
What is a simple mastectomy versus a radical mastectomy?
A simple (total) mastectomy removes the entire breast including skin and nipple with areola. A radical mastectomy includes the removal of axillary lymph nodes. These can be unilateral or bilateral.
121
What is an axillary lymph node dissection?
Axillary (underarm) is the traditional lymph node dissection. Examination of the lymph nodes in this area (twenty to thirty) is to determine whether cancer has spread to lymphatic system and, if so, staging the cancer by observing how many nodes are involved as well as things like size and shape.
122
What is the difference between an axillary lymph node dissection and a sentinel lymph node dissection?
A sentinal lymph node dissection is a tissue sparing procedure where a surgeon injects thd patient with a radioactive dye that follows the lymph drainage into nodes around a tumor. Only the closest nodes are dissected. If these sentinel nodes are not involved it is unlikely that other regional nodes are.
