11_Processing III11.pptx

Tissue Processing III Tissue Processors  Tissue Processors  Closed / Fluid transfer most popular today Pumps each solution in and out of retort Allows temp & pressure option for each station  Tissue Processors  Microwave processors are beco...

Tissue Processing III Tissue Processors  Tissue Processors  Closed / Fluid transfer most popular today Pumps each solution in and out of retort Allows temp & pressure option for each station  Tissue Processors  Microwave processors are becoming less popular Complete fixation reagents manually moved Isopropanol used as ‘intermediary” Wax is non-polar, no microwave attraction Allows temp & pressure option for each station Microwave Closed  Tissue Processing  May also be done by hand Extremely urgent specimens Possible prion infection Special circumstances  Tissue Processors  New automated instruments may achieve full processing in less than one hour Sakura Express or Tissue Tek Express Leica ASP300 Lecia Histocore Peloris  Processor Protocols  Fluid transfer-set to allow embedding first thing in the morning.  Microwave- possibly at intervals during the day. 2-3 runs  Continuous throughput instruments- new batch every 40 minutes.  Processor Protocols Fluid transfer  Tissue in the cassettes are ready to be brought to the embedder first thing in the morning  The weakness?  ( Tissues are all processed the same way, regardless of size or type)  Processor Protocols Protocols allow the operators to schedule  Time  Pressure  Heat Prolonged heat can cause tissue hardening  Processor Protocols fluid transfer processor  37°C  Vacuum  Processor can start immediately or delay by holding tissues in the first station  Microwave processing  Generally faster than fluid transfer  Requires careful design to ensure uniform heating  Processor Maintenance Set within laboratory Use manufacturer’s recommendations to develop SOPs  Changing reagents  When? age of reagents # of blocks run with reagents Any obvious contamination  Example cloudy xylene  Full change or rotation  See lab SOPs  Why does alcohol on a tissue processor require frequent changing?  Alcohol absorbs water from the tissues and from humidity in the air as well.  Used processor reagents Series of reagents 1 2 3 Most Medium Least Carry-over Carry-over Carry-over  Rotated processor reagents 1 2 3 Reagent 1 Reagent 2 Reagent 3 With new reagent Medium Least New clean carry-over Carry-over reagent  Source of contamination (carry-over)  Alcohol Water from tissues in aqueous fixative Water from the air  Clearing agent Alcohol Water if the last alcohol contains more than 2%  Paraffin Clearing agent Alcohol if clearing agents are not changed  Changing reagents P P P P P P P P A A A A A A A A R R R R R R R R R R R R R R R R A A A A A A A A F F F F F F F F I I I I I I I I N N N N N N N N #1 #2 #3 #4 #2 #3 #4 #1  What is the rationale for changing paraffin wax bathes on the processor?  To avoid clearing agent contamination Other Infiltration Media Seldom used in routine clinical lab  Other Embedding Media  Carbowax- water soluble wax  Celloidin – nitrocellulose compound  Plastics – glycol methacrylate GMA  Epoxy resin  Carbowax water soluble wax Solid polyethylene glycol ADVANTAGES DISADVANTAGES  Will not infiltrate if tissue is  No dehydration or clearing extremely fatty  Lipids are not removed  Cannot be floated on water, use one of the following Potassium dichromate,  Some enzymes can be diethylene glycol demonstrated Gelatin in water, formaldehyde and wax  Must be stored in desiccated plastic bags  Celloidin Nitrocellulose compound ADVANTAGES DISADVANTAGES  Perfect dehydration is required- will tolerate no water  No heat used in Closed containers alcohol & processing anhydrous ether Allows very delicate tissues to be processed  Process may take weeks to without distortion months Gradual increases in Favored for brain concentration 2-14% research  Blocks must be stored in 80% alcohol Celloidin blocks  Plastics – GMA an acrylic resin miscible in water ADVANTAGES DISADVANTAGES  Difficult microtomy  Embedding of calcified bone  Staining is difficult  Allows 1-2 µm sections  Dangerous chemicals, use  Great cellular detail under hood Kidney, bone marrow & lymph node  GMA KIT Block  Epoxy Resin- Araldite, Epon, Spurr  Used for electron microscopy  Diamond knife, 60-90 nm thick sections  Dehydrate and use transitional fluid such as propylene glycol if resin is not miscible with alcohol  Avoid skin contact Miscellaneous Infiltration  Agar and Gelatin  If many pieces of friable tissue will be made into a block, gelatin or agar may be used Block may frozen & used in cryostat Block may be embedded in paraffin  Friable- easily broken up Referred to as “double embedding”  Double Embedding  to combine the advantages of paraffin and nitrocellulose as embedding media  For cancellous bone or calcified tissues that may be crumbly 1. Infiltrate with celloidin (give more resiliency) 2. Infiltrate with paraffin and then embed in paraffin Double embedding  Frozen sections  30% sucrose solution A cryoprotectant to help protect tissue samples during freezing Prevents the formation of ice crystals, which can damage cell membranes and lead to artifacts  OCT (Optimal Cutting Temperature) compound Water-based compound composed of polyethylene glycol and polyvinyl alcohol. Provides a supportive matrix Troubleshooting Tissue Processing If fixation is optimal it is more difficult to harm a specimen while processing If fixation is not prompt or complete the damage is irreversible  Processor precipitation  Too high alcohol concentration following NBF  Zinc formalin must have pH below 7.0  Overdehydration Evidence - microchatter in tissue  Process biopsy and large specimens separately  Decrease dehydration time  Poor processing Evidence  Poor to absent nuclear staining  Water in tissue evident when placed in clearing agent  Is fixation adequate?  Poor processing  Change reagents more frequently  Change fume control as recommended  Condensation? Alcohols can absorb moisture and become dilute  Mechanical problem?  Use heat only for paraffin  Improper processing  Makes microtomy difficult. For example, mushy tissues. What could be a cause?  Decreases the quality of the slides produced  Failure to follow SOPs  Forgotten to load a solution  Loaded solutions in wrong sequence  Solutions not changed often enough  Programmed incorrectly Not defendable  When errors occur  Acknowledge  Attempt remediation  File incident report  How could this be prevented in the future? Open processors  Seldom used routinely  Have unique problems Troubleshooting cont. Handling other tissues  Handling small tissue samples FIXATIVE CONTAINING SMALL PIECE(S) OF TISSUE SMALL TISSUE PARTICLES  Sponge Artifact Use sponges soaked in fixative Use biopsy bags or lens paper  Specimen thickness  4mm or less in cassette  Garbage in-Garbage out- no processing method works if the sections are too thick for the solutions to penetrate.  Decalcification – covered on final, not quiz 3  5.15. Describe common decalcification methods utilized within the clinical environment with a particular focus on the following: o 5.15.1. Factors affecting decalcification o 5.15.2. Acid plus ion-exchange decalcification procedure o 5.15.3. Chelating agent decalcification procedures o 5.15.4. Advantages and disadvantages of each decalcification procedure o 5.15.5. Effect of decalcification on staining  5.16. Describe common procedures for endpoint detection  5.17. Discuss X-ray procedure with decalcification methods endpoint testing  5.18. Discuss the physical procedure with decalcification methods endpoint testing  5.19. Discuss CO2 production associated with decalcification methods endpoint testing  5.20. Discuss the advantages and disadvantages of each endpoint method  5.21. Describe cases that may require calcified bone sections  5.22. Describe procedures for section preparation of calcified bone sections, including: o 5.22.1. Adhesive tape method o 5.22.2. Sawing and grinding procedures o 5.22.3. Plastic embedding o 5.22.4. Techniques for softening hard tissue  5.23. Perform various essential tissue processing techniques, including: o 5.23.1. Dehydration o 5.23.2. Clearing o 5.23.3. Infiltration o 5.23.4. Paraffin processing o 5.23.5. Non-paraffin processing o 5.23.6. Decalcification Questions  Which is not a purpose of dehydration 1. Staining 2. Tissue Processing 3. Prolong the life of the tissue 4. Assist with coverslipping  The proper concentrations of alcohol for dehydration are descending. 1. True 2. False  Which of these is a dehydrant? 1. Xylene 2. Alcohol 3. Benzene 4. Formalin  Main disadvantage of xylene for clearing 1. Volatility 2. Tissue hardens 3. Poor miscibility with paraffin 4. Absorbs water  Which of these dehydrants requires the greatest volume? 1. Ethyl alcohol 2. Acetone 3. Methyl alcohol 4. Isopropanol  Most used dehydrant 1. Acetone 2. Ethyl alcohol 3. Isopropanol 4. Methyl alcohol  Most used clearing agent 1. Xylene 2. Acetone 3. Chloroform 4. Toluene  During staining, when is dehydration completed? 1. Before staining 2. After staining 3. During staining 4. Not performed  Clearing agents must be miscible with: 1. Paraffin and water 2. Fixative and dehydrant 3. Dehydrant and paraffin 4. Fixative and water  The tissue processor control panel is on fire. Your first response to this emergency should be?: 1. Pour some water over the control panel 2. Place a fire blanket over the processor 3. Cut the power supply to the processor 4. Evacuate the building  During microtomy, it is noted that many of the tissues examined seem very hard and shrunken. The most likely explanation? 1. Infiltrating paraffin is too hot 2. Processing reagents need changing 3. pH of fixative was incorrect 4. Clearing agent contaminated with water To speed processing of all surgical specimens; the temp for fixation, dehydration and clearing was set at 45°C. The likely result? 1. Excellent sections of all tissue 2. Very soft uterine scrapings 3. Overprocessed biopsy tissue 4. Sections will not stain with eosin Cloudy clearing agent may be contaminated with: 1. Absolute alcohol 2. Bacteria 3. Water 4. Yeast