Tissue Processing I & II PDF

Tissue Processing I & II  The Path of a Tissue Specimen Collection (OR, Clinic, Dr’s Pathology Lab Office) Accessionin Grossing Processing Embedding Microtomy Staining Assigning g...

Tissue Processing I & II  The Path of a Tissue Specimen Collection (OR, Clinic, Dr’s Pathology Lab Office) Accessionin Grossing Processing Embedding Microtomy Staining Assigning g Report Pathologist  What are the steps to tissue processing?  Fixation Must be complete before processing begins If fixation is not complete add fixation time to your processing schedule  Dehydration  Clearing  Infiltration  Embedding  Processing Tissue Processing – Tissue Processing aims to remove water from tissues and replace them with a medium that solidifies to allow thin sections to be cut Three Major Steps > Dehydration - Removes water from the tissue > Clearing - Removes alcohol from the tissue > Infiltration - Infiltrates the tissue with Paraffin wax Dehydration  Dehydration  Removes water from tissue Hydrophilic reagents attract water from tissue Repeated dilution of aqueous tissue fluids  Most common agent?  Ethanol is most commonly used as a dehydrant  Dehydration usually uses gradual increases in concentration. WHY?  50% - 60% - 70% - 80% - 90% - 100% (absolute)  Dehydration  When do you dehydrate? Tissue Processing  Removal of water  Intermediate step between aqueous and non-aqueous medium  Why? Water does not mix with infiltrating or embedding media (paraffin, celloidin)  Dehydrating agent must mix with water (from fixative) and clearing agent  Alcohol is miscible with both water and clearing agents (e.g. xylene)  Dehydration  When do you dehydrate? Con’t Staining (routine staining)  Most stains are aqueous, and since water does not mix with resinous mounting media, we need to remove all water before proceeding to use mounting media. Therefore, dehydration is performed after staining.  Also, after cutting sections, in order to stain them in an aqueous stain, we must hydrate them. After Embedding/Cutting/Drying  After drying in oven, before staining: Deparaffinize slides (take wax off) Clear, xylene Hydrate, 100…70% alcohol  Stain  Dehydrate, 70…100% alcohol  Clear, Xylene  Cover slip, using resinous mounting material  Reasons for Ascending (Graded concentration in Dehydration)  70-95-100% Alcohol Minimizes: a. Shrinkage of tissue b. Hardening of tissue c. Distortion from turbulence and convection currents Differences in specific gravities of water and alcohol. Going from water directly to 100% can cause tissue layers to separate…distortion  Delicate Tissues  start with even lower concentrations of alcohol, for example: 35, 50, 60, 75, 95 x 3, 100%  This may need to be done manually and have a special program on the processor  Processing Microwave Dehydration > Can decrease the dehydration time by allowing only one step > May cause tissue distortion  Dehydration  Closed processors Start @ approx. 60%  Processing Common Dehydrating Reagents > Ethyl Alcohol (Most Common) > Isopropanol > Acetone > Methyl Alcohol > Denatured Alcohol  There are advantages and disadvantages to each of the dehydrating reagents  We will discuss Ethyl Alcohol  Ethyl alcohol PRO CON  Non toxic  Cost  Mixes well  Prolonged exposure causes  Little shrinkage shrinkage and hardening graded alcohols  Delicate tissues  Record keeping & Theft eyes and embryos prevention  Fast acting  Some dye extraction  Reliable  Safety - alcohols  Flammable  Drinking factors Ethyl Methyl 2 propanol  Violent reactions with oxidizing agents including silver nitrate  > 24% needs waste management  Processing  Alcohol Safety > Flammable - Usage, storage and disposal considerations > Can cause violent reactions with oxidizing agents > Intoxicating (ethyl) - Methyl and Isopropanol - Poisonous > Disposal - Concentration 24% or lower can go down the sink - Concentrations higher than 24% require waste disposal company  Isopropanol PRO CON  Penetration  No good for celloidin (nitrocellulose embedding)  Less shrinkage and hardening  Eosin is insoluble in  Less expensive  2- propanol  No booze factors  Mildly irritating to eyes, nose and throat  Good substitute for the favourite  Acetone PRO CON  Speed  Requires a greater volume  Cost  Flammable Low flash point  Does not remove dye from  Excessive shrinkage stained sections  Best graded with xylene  Volatile – evaporates  Melts plastic coverslips  Methyl alcohol  Works well   May cause blindness or death  Denatured alcohol  Works well   Not subject to alcohol taxation  Security measures not needed  Record keeping avoided  Dehydration  Verify alcohol content Add to xylene-water turns white in xylene Check specific gravity to determine water content  Alcohol may be desiccated with calcium or copper sulfate Except….  Added to dehydrant  Softeners –phenol, mollifex (glycerin/alcohol mixture) or dish detergent in DI water. Helps preserve the flexibility of the tissue for better sectioning  Dyes – eosin to aid embedding  Processing Common Clearing Reagents > Xylene (Considered one of the best) > Toluene > Benzene > Chloroform > Acetone > Essential Oils (ex. Cedarwood oil) > Xylene Substitutes (ex. Limonene, Clearite)  There are advantages and disadvantages to all clearing reagents  They are all very toxic  Universal Solvents One reagent achieves both dehydration and clearing Advantage is speed Disadvantages are toxicity, cost & unpleasant aroma DIOXANE, TERTIARY BUTANOL or TETRAHYDROFURAN What two processing reagents must a universal solvent be miscible with?  Fixative (usually aqueous)  Paraffin Clearing ( closed processor) Dealcoholization of tissues and slides  Clearing  Removing the alcohol from the tissue & replacing it with a clearing agent dealcoholization  Must be miscible with: Dehydrating alcohols Infiltrating media such as paraffin Mounting media such as Permount (Slides)  Clearing agents  Many change the refractive index of tissue  Most clearing agents cause a problem if left in contact with tissue too long.  Incomplete clearing prevents paraffin infiltration and mushy blocks result  Why ? Poor infiltration  Criteria for Choosing Clearing Agents  Speedy removal of dehydrant  Ease of removal by molten wax  Minimal tissue damage  Flammability  Toxicity  Cost  Xylene advantages  Speed  The end is clear  Flexibility - Celloidin is not dissolved  No aniline dye removal  Cloudy with water  Well known  Disadvantages  Toxic  Intolerant – makes tissues hard  Hazardous waste Solvent recycle Licensed waste disposal  Xylene safety  Ventilation  Gloves  Flammable  Automation of coverslipping reduces exposure  Toluene PRO CON Less hardening of tissue Toxicity   especially with nerve and brain  Clear endpoint  Headache and dizziness  Speed  Dermatitis  Considered best by many folks  Lung injury  Long term exposure harmful to vital organs  Benzene advantages  Speed  Clear endpoint  Hardens >toluene but Low in toxicity (compared to others) > Gentle on tissue  Xylene Substitute (Clearite) - Disadvantages > Intolerant to water > Humidity may cause water contamination > Not compatible with all mounting medias (cover slipping) > Require more stations on processor than Xylene (3 vs 2)  Infiltration A supporting media which holds cells & intercellular structures in proper relationships while thin sections are cut.  Infiltration Infiltration > Infiltration media is added to the tissue to add support > Helps hold the cells and intracellular structures in proper formation during the cutting stage Common Infiltration Medias > Paraffin Wax - Most common - Melted and kept at 600C > Water soluble wax > Celloidin > Plastics > Epoxy resin  Infiltration media  Paraffin  Water soluble wax - carbowax  Celloidin – Nitrocellulose compound  Plastics – glycol methacrylate GMA  Epoxy resin  Paraffin  Cheap  Easily handled  Relatively easy section production  Shorter processing time  Paraffin  Allows serial sectioning  Allows many staining procedures  May be stored immediately after cutting  Safe to handle  Paraffin Additives change the properties  Beeswax – increases stickiness  Rubber – reduces brittleness  Other waxes – smooth texture, smaller crystals  Plastic - increases hardness and support  Proprietary Paraffin  An increased melting temperature indicates a harder wax formula Harder waxes allow thinner sectioning Ribboning is difficult  Decreased temperature – softer wax Better Ribboning, harder to get very thin sections & less tissue support  Paraffin  Most labs use the lower melting point (55- 58°C)  Paraffin on the tissue processor  Keep it short  potential for damage  Monitor temperature  Avoid clearing agent contamination  Paraffin under pressure  Speeds the process  Removes residual air  May damage tiny biopsies  Do not exceed 500mm Hg  Vacuum reduces infiltration time  Processing Tissue Processors (Open vs Closed)  Opened Processor - “Dip and Dunk” processor automatically moves tissue cassettes to various reagent containers - Requires more time - More hazardous - Errors may occur (ex. Dipped in wrong reagent, cassettes left out of reagent)  Processing  Closed Processor - Most common - Fluid transfer technique - Solutions are pumped in and out of the retort (holds cassettes) - Vacuum and heat can be applied  Processing  Microwave Processor - Decreases processing time - Requires complete fixation prior to processing - Reagents are manually added and removed - Isopropyl alcohol is an intermediate used instead of clearing agent  Manual/ Hand Processing > May be used for extremely urgent processing > Used for special circumstances > May be required for high-risk specimens (ex. Prions)  Processing  Processor Protocols  Generally, processors are programmed so that the tissue in the cassettes are ready to be embedded first thing in the morning (set finish time)  Protocols allow the operator to schedule the length of time the tissues are in each solution and whether pressure and/or heat is applied  Protocols may depend on tissue types and lab protocols  Processor can start immediately or have a delayed start  Delayed start involves a holding station as the first station (formalin -continued fixation)  Cleaning schedules/protocols must be followed  Processing  Sample Protocol (Fluid Transfer Processor)  Processing  Routine for changing reagents “prolong their  life” The first station of each reagent will be the most contaminated (ie. The first alcohol will have the most water, the first clearing agent will have the most alcohol, the first paraffin will have the most clearing agent)  Discard the most contaminated station and rotate the others  Fresh reagent will be placed in the last position Ex. Paraffin Wax Stations X 4 - Position 1 - discarded - Position 2 - moved to position 1 - Position 3 - moved to position 2 - Position 4 - replaced with fresh paraffin  Processing  Possible Contaminants  Alcohol Stations - Contaminated with water from the tissue or the air  Clearing Stations - Contaminated with alcohol (carryover) or water  Paraffin Stations - Contaminated with clearing agent (carryover) or alcohol ** If Xylene (clearing agent) is contaminated with water it will appear milky/cloudy ** If Paraffin is contaminated with clearing agent, it will have a strong odor