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Processing
Processing in the histology lab
| Question | Answer |
|---|---|
| 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
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