Grossing, Processing, and Instrumentation

Question 1

Could limonene be substituted for xylene as a clearing reagent?

Answer 1

Yes. it is a xylene substitute.

Question 2

Why is limonene not used more than xylene as a clearing reagent in laboratories.

Answer 2

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.

Question 3

Define dehydration in terms of tissue processing.

Answer 3

The removal of free water from tissue in order to embed in a non-aqueous medium. This is usually done with graded alcohols.

Question 4

What is clearing in tissue processing?

Answer 4

The removal of the dehydrating reagent (usually alcohol) in order to replace it with the infiltration medium (usually paraffin).

Question 5

Why must we infiltrate tissue with the embedding medium prior to embedding?

Answer 5

The embedding medium must permeate the tissue in order to keep it in place in the block during microtomy.

Question 6

True or False: Tissue can remain in hot paraffin indefinitely.

Answer 6

False: Tissue should remain in hot paraffin for the MINIMUM amount of time necessary for infiltration. This is to prevent heat damage.

Question 7

What is the correct temperature of molten paraffin in the tissue processor?

Answer 7

2° to 4° above the melting point of the paraffin.

Question 8

What is the optimal temperature range of the microtomy waterbath.

Answer 8

5° to 10° below the melting point of the paraffin.

Question 9

What is the most popular medium for tissue embedding?

Answer 9

Paraffin

Question 10

What is embedding?

Answer 10

Encasing tissue into a block that can be cut thinly on a microtome.

Question 11

What do histotechs mean by universal solvent?

Answer 11

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.

Question 12

What happens if formalin salts get into patient tissue?

Answer 12

Shredding during microtomy.

Question 13

What do histotechs mean by formalin salts?

Answer 13

The buffering salts used in 10% NBF. (Usually sodium phosphate monbasic and sodium phosphate dibasic)

Question 14

Why is the warm water flush done on the first 3 to 5 stations of the tissue processor.

Answer 14

The buffers used in 10% NBF are soluble in water but precipitate out in alcohol. Flushing prevents these salts from accumulating in the tubes.

Question 15

Can formalin salts precipitate onto the tissue as well?

Answer 15

Yes, if the first alcohol after formalin is 95% or higher, the phosphate buffers can precipitate onto and into the tissue.

Question 16

Name 3 dehydrating agents.

Answer 16

All alcohols, Acetone, Tetrahydrofuran, Dioxane.

Question 17

Name 3 clearing agents.

Answer 17

Xylene, Toluene, Tetrahydrofuran, Dioxane, Limonene, Benzene, Chloroform, Cedarwood Oil, Clove Oil, and Aliphatic Hydrocarbons.

Question 18

Name 3 universal solvents.

Answer 18

Acetone, Dioxane, Tertiary Butanol, and Tetrahydrofuran (or THF).

Question 19

How is the term cross-section used when embedding?

Answer 19

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.

Question 20

When would a grosser submit tissue for decalcification?

Answer 20

After fixation in order to remove calcium for paraffin embedding.

Question 21

Why is FFPE (formalin-fixed, paraffin embedded) tissue submitted for decalcification after fixation?

Answer 21

The tissue will continue to deteriorate during decalcification.

Question 22

If tissue for paraffin embedding is submitted for decal after fixation, when is tissue meant for embedding in plastic decaled?

Answer 22

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.

Question 23

Name 3 methods of decalcification.

Answer 23

Acid, Ion-exchange resins, electrolysis.

Question 24

What is the primary microscope used in the histopathology laboratory?

Answer 24

The light microscope.

Question 25

A compound microscope contains 2 magnifying lenses. What are they called?

Answer 25

Objective and ocular.

Question 26

Name some of the objective lenses and give their magnifications.

Answer 26

Scanning (2.5X to 4X), intermediate (10X to 20X), high-powered dry lens (40X to 45X), and oil immersion lens (90X to 100X).

Question 27

What is resolving power?

Answer 27

A measurement of the least distance between 2 objects that allows them to be SEEN AS 2 objects.

Question 28

What is the resolving power of the light microscope?

Answer 28

0.2 microns.

Question 29

How much are ocular lenses magnified?

Answer 29

5X to 15X.

Question 30

How do you compute the magnification of a particular slide under a compound microscope?

Answer 30

By multiplying the ocular lens magnification by the objective lens magnification.

Question 31

Agitation can be used on open and closed processors as well as manual processing. Why would we want it?

Answer 31

It facilitates fluid exchange.

Question 32

When 10%NBF is used, why should dehydration begin with an alcohol less than 70% concentration?

Answer 32

Because when fixing with zinc formalin or 10% NBF, precipitate can form in the processor tubing and/or patient tissue.

Question 33

How should bone be embedded?

Answer 33

Diagonally so that the microtome blade contacts only a small surface area of the tissue at a time.

Question 34

Describe 2 applications for a microwave oven in the histology laboratory.

Answer 34

Fixation, processing, and staining.

Question 35

How are tubes embedded?

Answer 35

On end.

Question 36

What is the best way to embed tissue with layers?

Answer 36

On edge.

Question 37

Name two types of electron microscopes.

Answer 37

Scanning and transmission.

Question 38

What are the three types of solidification?

Answer 38

Polymerization (paraffin and carbowax), Evaporation (celloidin), and Crystallization (resins and plastics).

Question 39

What is freeze artifact?

Answer 39

Holes in the tissue where ice crystals formed.

Question 40

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?

Answer 40

Eosin is insoluble in isopropyl alcohol. And Eosin IS 1/2 of our go-to stain.

Question 41

What is birefringence?

Answer 41

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.

Question 42

What is plane of section?

Answer 42

The position of the microtome cut in relation to the anatomical structures in the tissue.

Question 43

What is tissue processing?

Answer 43

The removal of free water from tissue through a series of graded alcohols in order to replace it by an infiltrating media.

Question 44

Why do we need to “fix” and”process” the tissue before staining it?

Answer 44

The goal of both tissue fixation and processing is to make the tissue firm enough for thin sectioning without disturbing the morphology.

Question 45

What is a microscope with only one lens called?

Answer 45

A simple microscope rather than a compound microscope that has two lenses. Aka a magnifying glass.

Question 46

After fixation with NBF, what concentration should the 1st alcohol be?

Answer 46

No more than 65%.

Question 47

How is a polarizing microscope used in the histology laboratory?

Answer 47

To view urate crystals, amyloid stained with Congo Red, and fixation pigments.

Question 48

What is a hydrometer?

Answer 48

An instrument used to measure the water content in alcohol.

Question 49

What is micrometry?

Answer 49

Microscopic measurement using an eyepiece and a stage micrometer.

Question 50

What is the difference between bright field and dark field microscopy?

Answer 50

Bright field shows images on a light background, and dark field shows images on a dark background.

Question 51

True or False: An ultrasection is a paraffin section with no wrinkles.

Answer 51

False. An ultrasection is a very thin tissue section cut on an ultramicrotome for EM.

Question 52

True or False: You can write on both cassettes and slides with a pencil, and it won’t come off during processing or staining.

Answer 52

True

Question 53

Name 3 factors that affect processing speed.

Answer 53

Agitation, Heat, Vacuum, Viscosity of Reagents.

Question 54

When decalcifying using acids, what is the ratio of reagent to tissue?

Answer 54

20:1, the same as it is for fixation.

Question 55

True or False: All acid decalcifiers affect tissue staining.

Answer 55

True. pH is very important in staining, and all acids affect staining to some degree.

Question 56

Is it possible to decalcify with weaker acids?

Answer 56

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)

Question 57

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?

Answer 57

It does. That’s why you must change the decalcifier often.

Question 58

Do agitation, vacuum, and heat help with decalcifying as they do with fixation and processing?

Answer 58

Agitation and vacuum are great, but heat is a no-no when decalcifying. Actually, heat should be used cautiously in fixation and processing.

Question 59

What is considered the best method for decalcifying bone?

Answer 59

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.

Question 60

How does ion exchange work on decalcification?

Answer 60

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.

Question 61

The electrolytic method of decalcification works within a few hours. Why don’t most labs decalcify with this method?

Answer 61

The heat pretty much destroys the tissue.

Question 62

What’s the decalcifying agent that uses chelation?

Answer 62

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.

Question 63

What is meant when we say tissue was underprocessed?

Answer 63

Enough of the free water was not removed, causing incomplete clearing and infiltration.

Question 64

What is meant by overprocessing tissue?

Answer 64

Biologically bound water was removed during processing, causing microchatter.

Question 65

What is the magic percentage of alcohol for tissue storage?

Answer 65

70%. This is isotonic for tissue storage. Lower and the tissue will swell. Higher and the tissue will shrink.

Question 66

Why is reagent alcohol not used in the EM lab?

Answer 66

Methanol content. Same reason EM labs don’t fix with formalin.

Question 67

Most histo labs add eosin to one of the last dehydrating alcohols. Is that a good idea?

Answer 67

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.

Question 68

Is Anatech’s Pro-Soft a good dehydrant for biopsies?

Answer 68

Yes, it is. It doesn’t remove bound water and works well with the Peloris tissue processing method.

Question 69

Why is clearing called clearing?

Answer 69

Clearing reagents have the same refractive index as tissue, so it looks translucent.

Question 70

What step in tissue processing could cause damage to the tissue.

Answer 70

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.

Question 71

Is Xylene an Aromatic Hydrocarbon?

Answer 71

Yes, so are Benzene and Toluene (think smelly). Not to be confused with Aliphatic Hydrocarbons, which are xylene substitutes.

Question 72

Can chloroform be used as a clearing reagent?

Answer 72

Yep.

Question 73

Can you store tissue in Essential Oils, such as Cedarwood Oil, Clove Oil, Sandalwood Oil, and Wintergreen Oil?

Answer 73

Yes. You would use the essential oil for clearing and just not infiltrate them until needed.

Question 74

Name 3 infiltrating mediums.

Answer 74

Paraffin, Carbowax, Celloidin, Plastic

Question 75

What do Beeswax, Rubber, Bayberry Wax, and Plastic have in common?

Answer 75

They are all paraffin additives.

Question 76

What is Polyethylene Glycol?

Answer 76

Carbowax, a water soluble wax.

Question 77

True or False: Celloidin hardens through crystallization.

Answer 77

False. It solidifies using evaporation.

Question 78

How do plastics and resins solidify?

Answer 78

Crystallization.

Question 79

What is the most common microtome for FFPE (formalin fixed paraffin embedded) tissue?p

Answer 79

The rotary microtome.

Question 80

What are the most obvious differences between a rotary microtome and a sliding microtome?

Answer 80

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.

Question 81

What type of knives are used in EM?

Answer 81

Glass and Diamond.

Question 82

What type of knife is used for plastic embedded tissue?

Answer 82

A Ralph (glass) knife.

Question 83

Nowadays, knives have been replaced by disposable blades. Do they come in different sizes?

Answer 83

Yes, high and low profile.

Question 84

On a rotary microtome, what is the correct clearance angle between the bevel of the blade and the block face?

Answer 84

2-5°

Question 85

What can happen during microtomy when the clearance angle between the blade and the block is too slight? (Blade tilt is too verticle)

Answer 85

Skipped sections, thick and thin, wrinkles

Question 86

What can happen during microtomy when the clearance angle between the block and the blade is too large? (Blade tilt too horizontal)

Answer 86

Chatter, microchatter, washboarding, trouble getting a ribbon

Question 87

Why should the floatation, or hot water, bath be filled with hot water?

Answer 87

It helps keep it free of air bubbles.

Question 88

True or False: The hot water bath surface needs to be cleaned after every block.

Answer 88

True.

Question 89

True or False: Waterbaths should be cleaned once a week.

Answer 89

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.

Question 90

True or False: Slides go in the oven before staining in order to melt the paraffin before deparaffinization.

Answer 90

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.

Question 91

What happens when the slide drying oven temperature is too hot?

Answer 91

Darkened nuclei, nuclear bubbling, loss of nuclear detail.

Question 92

What happens when the slide drying oven temperature is too low?

Answer 92

Tissue falls off slides because they were not completely dried.

Question 93

How can you be sure the slide drying oven is at the right temperature?

Answer 93

Check the melting point of the paraffin. Should be less than 70°.

Question 94

When should you choose to use charged slides (or slides with adhesives)?

Answer 94

When staining solutions are alkaline, for frozen sections, for IHC or IF, with decals, brains, or under processed tissue.

Question 95

What are slide adhesives, and why do most labs no longer add adhesive to the waterbaths?

Answer 95

Gelatin, Poly-L-lysine, Silane, Albumin. They tend to cause background staining.

Question 96

What are the three main mechanical causes of microtomy sectioning difficulties?

Answer 96

Dull blade, wrong clearance angle, something loose.

Question 97

What can you do if you can’t get a ribbon?

Answer 97

Blow on the block to warm it up or run a dowel stick over the blade to dull it a little.

Question 98

Why might you see compression in your ribbons?

Answer 98

Dull blade, hard tissue, incomplete infiltration.

Question 99

What should you do if your ribbons are compressed?

Answer 99

Get a new blade and make sure the block is cold.

Question 100

What happens when the bottom off the block is not parallel to the blade?

Answer 100

The ribbon will curve or slant.

Question 101

What happens when you rough your blocks too aggressively and don’t “polish”?

Answer 101

Holes in the tissue. Fix by polishing the block.

Question 102

When sectioning, why would you get a knife line?

Answer 102

A nick in the blade, debris in the paraffin, calcium, keratin, sutures, staples.

Question 103

What is parched earth?

Answer 103

Cracks in tissue sections due to dryness.

Question 104

What is the specific name for the person performing an autopsy or dissecting an organ.

Answer 104

The prosector. More commonly called the grosser. Sometimes called the cutter, although that term is also used for microtomists.