Histo

• Fixation


  Tissues placed in fixative solution to preserve morphology, usually via stabilization of protein
• Fixation


  • May be started before or after dissection
  • Small biopsies often arrive in small bottles of formalin
  • Fresh O.R. specimens need to be fixed in the grossroom prior to processing
  • Bone specimens need to be decalcified after fixation, before processing

  
• Fixation must be complete before tissue processing
• The size of a piece of tissue determines how long to both fix and process for
• It is important not to overfill cassettes when grossing as this will cause distortion or poor processing
• Goal of tissue processing

  To remove H2O and have complete wax impregnation of tissue so that it may be embedded within a block of paraffin wax

  
• Steps of Tissue Processing

  1. Dehydration

  2. Clearing

  3. Impregnation
• Dehydration


  To remove water and fixative solutions from tissue and replace them with dehydrating solutions
• Dehydrating solutions


  • Ethanol
  • Methanol
  • Isopropyl alcohol
  • Acetone
• Ethanol


  Miscible with water and most of the commonly used dehydrants, but even absolute ethanol contains some water (1-2%) which can result in inadequate dehydration
• Ethanol


  • Expensive due to government duties
  • Government mandated record keeping
  • Over exposure results in tissue hardening
  • Removes lipids
  • Flammable and toxic
• Isopropyl alcohol


  Miscible with water, ethanol and clearing agents, less expensive than ethanol, does not harden or shrink tissue as much as ethanol, somewhat less toxic than ethanol and removes fewer lipids
• Methanol


  Toxic, liver converts it to formaldehyde which can cause acidosis, tissue damage or blindness, less expensive than ethanol
• Acetone


  Slow to penetrate therefore takes longer, causes excessive shrinkage and brittleness, removes lipids, absorbs water from air therefore must be kept in airtight containers
• Under-dehydration


  Residual water prevents adequate penetration of xylene and wax, caused by too little time in dehydrant or contamination of last alcohol bath, results in soft, mushy blocks that may smell of alcohol and have large holes in the tissue

  
• Remedy for under-dehydration


  Reprocess blocks by melting, removing wax with xylene, returning to new absolute alcohol, then reprocessing with fresh xylene and wax
• Over-dehydration


  Tissues become too hard and brittle, may not be able to cut the block
• Clearing


  Dehydrating fluids are not miscible with wax therefore need an intermediate clearing agent that is miscible with both alcohols and wax
• Clearing agents


  • Xylene
  • Xylene substitutes
  • Chloroform
  • Benzene
• Xylene


  Very common, rapid, may harden tissue, not recommended for some tissues, strong odor, may cause headaches, dizziness, respiratory issues with prolonged exposure
• Xylene substitutes


  Variable toxicities, some less effective at penetration or wax removal, generally expensive, increasing in popularity due to safety concerns with xylene
• Benzene/Toluene


  Similar in action to xylene, benzene is very toxic and a carcinogen in humans, requires ventilation and sealed containers, not usually used in routine labs
• Chloroform


  Makes tissues less brittle than xylene, penetrates tissues very slowly, hygroscopic (absorbs water), extremely toxic, may cause convulsions, coma and death, does not make tissues transparent, rarely used
• Impregnation


  Purpose is to replace clearing agent with impregnating/embedding medium, paraffin wax is the most popular medium
• Paraffin wax


  Usual melting point 56-58°C, kept molten in baths/reservoirs at ~62°C, crystalline substance that reforms as wax cools and hardens, rapid cooling is optimal
• Paraffin wax


  • Inexpensive
  • Readily available
  • Sections easily on the microtome once hardened
  • Inert, allows for long-term storage of blocks
• Wax additives


  May be used to slightly alter wax properties, plastic polymers support improved tissue infiltration and allow for quality sections with minimal compression
• Phosgene gas


  Gas given off when paraffin wax is heated, has been used as a weapon
• Phosgene gas


  • May cause convulsions, coma and death due to respiratory failure or cardiac arrhythmias
  • Unlike xylene, it does not make tissues transparent
  • Rarely used
• Impregnation


  Purpose is to replace clearing agent with impregnating/embedding medium
• Paraffin wax


  • Most popular impregnating medium
  • Inexpensive
  • Readily available
  • Sections easily on the microtome once hardened
  • Inert; allows for long-term storage of blocks
• Paraffin wax


  • Usual melting point (in North America) 56 – 58°C, kept molten in baths/ reservoirs at ~62°C
  • Crystalline substance
  • Crystals reform as wax cools and harden
  • Rapid cooling is optimal
• Wax additives


  • Plastic polymers support improved tissue infiltration and allow for quality sections with minimal compression, promoting wrinkle-free sections
  • A small percentage of dimethyl sulfoxide (DMSO) allows for accelerated tissue penetration
  • Some additives can create higher melting points in wax for use in warmer climates
• Other impregnating media


  Epoxy glycol methacrylate used when tissue needs to be cut extremely thin (2 microns or less), e.g. for electron microscopy

  
• Enhancing tissue processing


  1. Movement of processing fluids to increase rate of exchange of fluids through and between tissue

  2. Fluids pumped in and out of reservoir to facilitate movement

  3. Agitation of tissues

  4. Increased temperature increases rate of penetration of fluids

  5. Vacuum can be applied to remove and add fluids to increase rate of wax impregnation
• Tissue processor


  • Automated machine that performs all the steps of processing
  • All of the fluids are in bottles or containers
  • The duration tissues are in each solution may be programmed
  • A complete tissue processor program is often referred to as a "run"
• Types of automated tissue processors

  • Fluid Transfer / Fluid Exchange: Tissue cassettes stay in place while reagents pumped in and out
  • Tissue Transfer: Tissue cassettes move from reagent to reagent
• Tissue transfer processor


  • Vapours not enclosed
  • Less expensive
  • Difficult to control vacuum and heating
  • Harder to organize blocks
  • Quick evaporation of reagents
  • Not commonly used in high volume diagnostic histology laboratories
• Enclosed fluid exchange processor

  • Modern processor with a completely enclosed vessel where tissue cassettes are placed
  • Vapours enclosed within system and filtered
  • Tissues do not dry out
  • More complex and expensive
  • Consistent vacuum and heat
  • Minimal evaporation of reagents
• Processing schedules


  1. Formalin (0-2 hours, 1-2 bottles)

  2. 70% Ethanol (1 hour, 2 bottles)

  3. 95% Ethanol (1 hour, 2 bottles)

  4. 100% Ethanol (1 hour, 3 bottles)

  5. Xylene (1 hour, 3 bottles)

  6. Wax (1.5 hours, 3 bottles)