Grossing, Processing, and Instrumentation Flashcards
Could limonene be substituted for xylene as a clearing reagent?
Yes. it is a xylene substitute.
Why is limonene not used more than xylene as a clearing reagent in laboratories.
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.
Define dehydration in terms of tissue processing.
The removal of free water from tissue in order to embed in a non-aqueous medium. This is usually done with graded alcohols.
What is clearing in tissue processing?
The removal of the dehydrating reagent (usually alcohol) in order to replace it with the infiltration medium (usually paraffin).
Why must we infiltrate tissue with the embedding medium prior to embedding?
The embedding medium must permeate the tissue in order to keep it in place in the block during microtomy.
True or False: Tissue can remain in hot paraffin indefinitely.
False: Tissue should remain in hot paraffin for the MINIMUM amount of time necessary for infiltration. This is to prevent heat damage.
What is the correct temperature of molten paraffin in the tissue processor?
2° to 4° above the melting point of the paraffin.
What is the optimal temperature range of the microtomy waterbath.
5° to 10° below the melting point of the paraffin.
What is the most popular medium for tissue embedding?
Paraffin
What is embedding?
Encasing tissue into a block that can be cut thinly on a microtome.
What do histotechs mean by universal solvent?
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.
What happens if formalin salts get into patient tissue?
Shredding during microtomy.
What do histotechs mean by formalin salts?
The buffering salts used in 10% NBF. (Usually sodium phosphate monobasic and sodium phosphate dibasic)
Why is the warm water flush done on the first 3 to 5 stations of the tissue processor.
The buffers used in 10% NBF are soluble in water but precipitate out in alcohol. Flushing prevents these salts from accumulating in the tubes.
Can formalin salts precipitate onto the tissue as well?
Yes, if the first alcohol after formalin is 95% or higher, the phosphate buffers can precipitate onto and into the tissue.
Name 3 dehydrating agents.
All alcohols, Acetone, Tetrahydrofuran, Dioxane.
Which is the faster dehydrant, Alcohol or Acetone?
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.
Name 3 clearing agents.
Xylene, Toluene, Tetrahydrofuran, Dioxane, Limonene, Benzene, Chloroform, Cedarwood Oil, Clove Oil, and Aliphatic Hydrocarbons.
Name 3 universal solvents.
Acetone, Dioxane, Tertiary Butanol, and Tetrahydrofuran (or THF).
How is the term cross-section used when embedding?
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.
When would a grosser submit tissue for decalcification?
After fixation in order to remove calcium for paraffin embedding.
Why is FFPE (formalin-fixed, paraffin embedded) tissue submitted for decalcification after fixation?
The tissue will continue to deteriorate during decalcification.
If tissue for paraffin embedding is submitted for decal after fixation, when is tissue meant for embedding in plastic decaled?
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.
Name 3 methods of decalcification.
Acid, Ion-exchange resins, electrolysis.
What is the primary microscope used in the histopathology laboratory?
The light microscope.
A compound microscope contains 2 magnifying lenses. What are they called?
Objective and ocular.
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).
What is resolving power?
A measurement of the least distance between 2 objects that allows them to be SEEN AS 2 objects.
What is the resolving power of the light microscope?
0.2 microns.
How much are ocular lenses magnified?
5X to 15X.
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.
Agitation can be used on open and closed processors as well as manual processing. Why would we want it?
It facilitates fluid exchange.
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.
How should bone be embedded?
Diagonally so that the microtome blade contacts only a small surface area of the tissue at a time.
Describe 2 applications for a microwave oven in the histology laboratory.
Fixation, processing, and staining.
How are tubes embedded?
On end.
What is the best way to embed tissue with layers?
On edge.
Name two types of electron microscopes.
Scanning and transmission.
What are the three types of solidification?
Polymerization (paraffin and carbowax), Evaporation (celloidin), and Crystallization (resins and plastics).
What is freeze artifact?
Holes in the tissue where ice crystals formed.
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.
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.
What is plane of section?
The position of the microtome cut in relation to the anatomical structures in the tissue.
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.
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.
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.
After fixation with NBF, what concentration should the 1st alcohol be?
No more than 65%.
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.
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.
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.
What is the shape of a Ralph knife when it is being used to cut ultrathin sections?
A triangle.
Nowadays, knives have been replaced by disposable blades. Do they come in different sizes?
Yes, high and low profile.
On a rotary microtome, what is the correct clearance angle between the bevel of the blade and the block face?
2-5°
What is parched earth?
Cracks in tissue sections due to dryness.
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.
What is a skin excision?
A segment of skin that has been surgically removed. Usually circular or elliptical, but can be irregular.
What does it mean to breadloaf a specimen?
To slice from tip to tip.
Why do we breadloaf skin excisions?
So the pathologist can examine the layers of skin.
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.
What is a deep margin?
The margin located at the base of the specimen. In skin this would be the subcutaneous soft tissue.
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.
Why are specimens inked?
To indicate orientation microscopically. In this way, the distance of a lesion to a margin can be measured and reported.
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.
