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Processing

Processing in the histology lab

QuestionAnswer
Dehydration Removal of water
Accounts for the vast majority of processing problems Incomplete dehydration
Dehydrating agents remove water in two ways 1. Some reagents are hydrophilic (water loving) and attract water from the tissue 2. Others dehydrate by repeated dilution of the aqueous tissue fluids.
Majority of dehydrating agents are Alcohols
Colorless biopsies can be hard to identify during embedding. What is added to alcohol to dye the tissue? Eosin or phyloxine
Drierite Indicates completeness of dehydration and dyes tissue for identification during the embedding step
Can be used as a dye with ethanol but not isopropyl Eosin and Drierite
The color in Drierite is cause from Cobalt chloride
Can't be used on enclosed processors to dye tissue Drierite
Hampers test results for FISH eosin and erythrocin B
FISH stands for Fluorescent in situ hybridization
Types of dyes that can be added during processing to add color to the specimen include Eosin, phyloxine, methylene blue, erythrocin B
Drinking alcohol Ethyl alcohol
Best of the dehydrants Ethyl alcohol
Hydrophillic dehydrant Ethyl alcohol
If phosphate buffered formalin is used for fixation then the tissue must be placed in alcohol solution of >_% or else the phosphate salts will precipitate and make it difficult to cut. 70%
PEL 1,000ppm and very flammable Ethyl alcohol
Must not exceed 24% solution when dumped down the drain Ethyl alcohol
Rarely used for dehydration alone Methyl alcohol
Unpleasent odor Methyl alcohol
Primary for the fixation of blood smears Methyl alcohol
Poisoness, broken down to formaldehyde by the liver and acts as formaldehyde on the body Methyl alcohol
Overexposure can cause blindness and death Methyl alcohol
PEL 200ppm and protective gear should be worn to prevent absorbtion Methyl alcohol
Cant be substituted in the preparation of staining solutions because it is insoluble Isopropyl alcohol
Cant be used for Celloidin because nitrocellulose is insoluble in it Isopropyl alcohol
Never pure, always contains some water Isopropyl alcohol
PEL 400ppm and toxic by ingestion Isopropyl alcohol
When dumped down the drain it should be at a concentration of <10% because it is considered ignitable at higher concentrations Isopropyl alcohol
Good for dehydrating when animal or plant tissue is involved Butyl alcohol
Pronounced odor and a low dehydrating power Butyl alcohol
PEL 100ppm Butyl alcohol
Rapid dehydrant and less expensive Acetone
When exposed to air this dehydrant will absorb water Acetone
Acetone Flashpoint -17 degrees Celsius
PEL 1,000ppm OSHA Acetone
PEL 250ppm NIOSH Acetone
Universal solvent Perform both dehydration and clearing steps
Miscible with water and with paraffin Universal solvents
Universal solvents include Dioxane, teritiary butanol, tetrahydrofuran
OSHA PEL 100ppm NIOSH PEL 1ppm Dioaxane (Universal solvent)
Expensive and solidifies at room temp Teritiary Butanol
PEL 100ppm Teritiary Butanol
Long term exposure can cause kidney or liver damage Tetrohydrofuran
PEL 200ppm and a STEL at 250ppm Tetrohydrofuran
Clearing agents must be miscible with what two substances? Dehydrating agent and the infiltrating medium
Most widely used clearing agent Xylene
Intolerant of water Xylene
Turns cloudy in the presence of water Xylene
May not be poured down the drain Xylene
PEL of 100ppm and STEL of 150ppm Xylene
Repeated exposure can cause CNS damage Xylene
Does not overharden tissue as much as Xylene does Toluene
May remain in this clearing agent over night with no harm Toluene
Greater tolerance for atmospheric water contamination than Xylene Toluene
PEL 50ppm and STEL 150ppm Toluene
Fast and does not over harden tissue as much as Xylene Benzene
Hardens muscle, uterus, and tendon more than Toluene Benzene
Very toxic Benzene
OSHA PEL 10ppm NIOSH PEL 0.1ppm Benzene
Carcinogen that affects primarily blood and bone marrow Benzene
Leaves tissue less brittle than Xylene and penetrates slow Chloroform
Better clearing agent for uterus, muscle, and tendon Chloroform
Does not make tissue transparent Chloroform
Not flammable or combustible Chloroform
Heating this clearing agent can form a toxic gas (phosgene) Chloroform
PEL 50ppm and a known carcinogen Chloroform
Very expensive and are only used for special projects Essential oils
This must be removed by a hydrocarbon in order to cut easily (Before infiltration) Essential oils
Essential Oils Sandalwood, clove, origanum, and cedarwood
Best known essential oil Cedarwood
Xylene substitutes Limonene reagents
Not water soluble, thus can't be dumped down the drain Limonene reagents
Who enforces the dumping of substances that are not miscible with water? EPA
Aliphatic hydrocarbons Alkanes
Less aggressive than Xylene Aliphatic hydrocarbons
Non irritating and nonsensitizing clearing agent Aliphatic hydrocarbons
Other clearing agents Carbon tetrachloride, Carbon bisulfide, and aviation gasoline
Holds the intracellular structures in their proper relationship while thin sections are cut Infiltrating medium
Most popular infiltrating medium Paraffin
What is paraffin made up of? Beeswax: reduces crystal size and increases stickiness rubber: reduces brittleness, increases stickiness and makes it easier to form ribbons other waxes: produce smooth texture and smaller crystal size
As the melting point of paraffin is raised the paraffin becomes _ and sectioning thinner sections becomes_ Harder and difficult
Typical melting point range of paraffin is what? 55-58 degrees Celsius
Overheating the embedding paraffin may change what? Sectioning quality
Over processing a biopsy will result in what? Hard tissue, difficult microtomy, and potentially affected staining
Water soluble waxes Carbowax
Because tissue can be embedded directly from an aqueous fixative to this water soluble wax it will not eliminate the fat and allow it to be seen Carbowax
Will not infiltrate large amounts of fat Carbowax
CNS requires longer periods of infiltration Carbowax
3 changes of wax for 3 hours is recommended Carbowax
This wax remains softer compared to paraffin Carbowax
Greatest problem encountered is when the sections float out on the water bath Carbowax
Best to cut these types of blocks when they have been chilled Carbowax
Nitrocellular compound used for embedding Celloidin
Any fixative may be used before the use of this infiltrating medium Celloidin
Blocks are hardened with chloroform Celloidin
Long tem storage of blocks requires storage in 80% alcohol Celloidin
Serial and thin sections are hard to obtain with this embedding medium Celloidin
Method is hazardous because anhydrous and nitrocellulose are used which are both explosive Celloidin
If _ types of strips break down into a crumbly powder, then a professional hazardous team may be needed for disposal Celloidin
Acrylic resin that is miscible with water Glycol Methacrylate
Provides excellent support for hard tissue Glycol Methacrylate
This type of knife allows hard sections to be cut thin Glass
Especially useful for kidney, bone marrow, and lymph nodes Glycol Methacrylate
Sections that are embedded in this medium tend to not adhere to the glass slide Glycol Methacrylate
Requires dehydration of the specimen and unless miscible with ethanol, they also require the use of transitional fluid Epoxy resins
Most frequent transitional fluid for epoxy resins Propylene Oxide
Most commonly used epoxy resins include... Araldite, Epon, and Spurr
Type of embedding medium needed when using electron microscopy or ultrastructural examination of the tissue is desired because it allows for thin sections to be cut Epoxy resins
With properly embedded tissue and a diamond knife, 60-90nm thick can be cut Epoxy riesins
0.5 micrometer sections, referred to as "thick" sections are used and cut with a glass knife light microscopy
Precipitate in the processor chamber & in the tubing Caused when phosphate buffered formalin is used for fixation and then dehydration is begun with 70% alcohol or higher. Also is caused when the pH of zinc formalin is >7.0
When precipitate occurs in the processor chamber and the tubing, what happens to microtomy Artifacts are seen within the tissue and it makes cutting difficult
How to clean the chamber and tubing after a precipitate has formed Rinse with diluted acetic acid (5-20%)
Chatter is seen when the specimen is overly _ Dehydrated
Poor processing is most likely noticed from poor nuclear staining
Most common reason of poor processing is When water remains in the tissue when being placed in the clearing agent
Sponges must be presoaked in fixative or else the specimen with show this pattern when staining the tissue Cross hatching or triangular patterns
Cysts, gallbladders, and gastrointestinal tracts must all be embedded how? On edge so that all layers are visible
Fallopian tubes, vas defrens, appendixes are embedded how? As a cross section so that the lumen and all layers of the mucosa, submucosa, and external muscle layers are obvious microscopically
Soft mushy tissue can be prevented by... 1. Ensuring that the tissue is not cut too thick 2. Tissue is allowed to fix for an adequate amount of time 3. Reagents are fresh/ changed on a regular basis
Incorrect orientation can be prevented by... 1. Marking the side of the tissue that needs to face up with ink 2. Notching the side of the tissue to be embedded up with a shallow "v" 3. Placing embedding instructions on the side of the cassette
Tissue carry over can be prevented by... 1. Cleaning the forceps with gauze in between specimens 2. Opening one cassette at a time
Tissue not embedded at the same level can be prevented by... 1. Pressing down on the tissue uniformly in the mold 2. Keeping the paraffin warm enough to embed everything on the same level 3. Working fast
Pieces of tissue missing from the block can be prevented by... 1. recording the number of pieces to be embedded 2.Double checking the number of pieces to be embedded 3. Checking the lid of cassette 4. Carefully opening paper and scraping
Decalcification is performed after _ and before _ After fixation and before dehydration
Acid methods of decalcification 1. Simple acids 2. Ion exchange 3. Electrolytic methods
Simple acids Calcium ions are allowed to migrate out of the tissue into the surrounding solution.
Examples of simple acids include hydrochloric acid and nitric acid
If hydrochloric acid is to be used to decalcify a specimen that has been fixed formalin then it must be washed first to prevent the formation of a carcinogen
Ammonium ions from the resin are exchanged for calcium ions Ion exchange resins
One of the best decalcification methods Ion exchange resins
Uses a mixture of formic and hydrochloric acids placed in an apparatus. The bone is attached to an anode (positive pole) and then a current is passed through the solution. Calcium ions are attracted away from the anode to the cathode. Electrolytic method
Takes about 2-6 hours Electrolytic method
Heat is used in this type of decalcifying method Electrolytic method
Chelating agents Organic compounds that have the property of binding certain metals.
EDTA binds _ calcium ions
End point decalcification 1. Mechanical methods 2. Chemical methods 3. Radiography
Mechanical method Bending, poking, scratching
Chemical method Dependent on the precipitate of calcium oxalate when a sample of decal fluid used is mixed with a solution containing ammonium hydroxide and ammonium oxalate. If the solution is turbid after 30 minutes then it has not reached proper end point
Radiography X-ray image
Radiography can't be used if the bone specimen was fixed in B-5, zinc formalin, or Zenker
Most accurate method of determining decal end point Radiography
How to prevent bone dust 1. Use a diamond blade 2. Trim bone surfaces after decal but before processing
How to prevent under decalcification 1. Choose a decal option with a fast turn around time 2. Choose a good method for determining end point 3. Check endpoint with radiography when in doubt
How to prevent over decalcification 1. Choose a decal agent that fits the needs of the lab 2. Choose a good method for determining end point 3. Check end point with radiography when in doubt
Frozen sections are best obtained by using Isopentane and liquid nitrogen
Isopentane should reach this temp before adding the tissue to freeze -150 degrees Celsius
If isopentane is not available then what can be done to the tissue to prevent the formation of gas bubbles Tissue can be dusted with Talc
Most satisfactory method for obtaining frozen sections from a formalin fixed tissue is Using an aqueous solution of 30% sucrose before freezing
How to prevent freezing artifact in frozen sections 1. Using a heat extractor 2. Freezing with isopentane chilled to -150 degrees Celsius 3. use other methods of rapid freezing 4. Ensuring tissue is not immersed in saline before freezing
How to prevent block loosening from the chuck while sectioning 1. reattach the tissue block to a clean chuck with additional embedding medium 2. avoid the storage of chucks without embedding medium in the cryostat 3.avoid the storage of chucks with embedding medium in the cryostat overnight during the defrost cycle
Most common reason the chuck detaches while sectioning Chuck was too cold when embedding medium was applied
How to prevent tissue not being flat when cutting in a cryostat Place tissue on a slide and apply embedding medium all around. As the embedding medium begins to turn white then coat a chuck with embedding medium and transfer the tissue that was flat on the slide to the chuck.
Created by: Ziek98