histopath lab mid

• Clearing


  • Removal of dehydrating agent itself from the tissue
  • Should be miscible with both the dehydrating agent and the embedding medium
  • Refractive index of the clearing agent is similar to the tissue
• Infiltration and Embedding

  1. Tissue space is now infiltrated with the embedding media

  2. The molten wax is solidified to provide support for cutting into thin section
• Ideal impregnating medium

  • Miscible with clearing agent
  • Liquid in higher temperature (30–60 °C) and solid in room temperature
  • Homogenous and stable
  • Non-toxic and cheap
  • Transparent
  • Fit for sectioning the tissue

  
• Different but has same medium except infiltrate cavities and remove clearing agent
• Melting point should not be higher because it can affect viscosity
• Lower temp=more viscous= problem with penetration
• Ideal temperature of paraffin wax -at least 2 degrees Celsius above to be easily observe
• Time duration and the number of changes

  • Size of tissue
  • Type of tissue
  • The type of clearing agent
  • Type of processing
• Automated - Tissue processor


  1hr each
• Can be reduced by 75% by using vacuum or liquid type tissue processing (machine uses negative pressure)
• Paraffin Wax


  • By-product during refining of crude petroleum
  • Melting point 39 °C to 70 °C
• Advantages of Paraffin Wax

  • Tissue block can be stored for long duration
  • Non-toxic
  • Cheap
  • Safe
• Disadvantages of Paraffin Wax

  • It may cause tissue shrinkage and hardening in case of prolonged impregnation
  • Long duration for the impregnation of the bone and eye
• To make hard wax add stearic acid
• To reduce melting point - add phenothionine
• To add more adhesiveness - add serosine
• For lung tissue - DMS remove excess clearing agent and purifies
• Copper sulfate - remove water from dehydrating agent, indicator for us to change alcohol, more water is absorbs = more blue color
• Tissue Processing Methods

  1. Manual

  2. Automated tissue processor - transfer the tissue in different fluid for a specified time in a desired environment
• Automated tissue processor

  • Tissue transfer processor - bucket of tissue is transferred from one carousel to other after a specified time
  • Fluid transfer processor - completely closed processor, exposed to fumes, specimen should have additive for every 5 mins, most used, well ventilated area, glass jar
• Incomplete dehydration - most common problem, solution: replace alcohol, 2 to 3 processing
• Advantages of Manual Tissue Processor

  • Small number of samples can be processed in a small laboratory
  • Careful monitoring in each step is possible
  • It is possible to select the reagents of choice with flexibility in time duration
• Disadvantages of Manual Tissue Processor

  • Inconvenient for processing and time taken procedure
• Microwave Processing

  • Suitable for small number of delicate tissues
  • Components: System to control the temperature, System to control the time duration of particular temperature, Proper exhaust to remove the toxic gas
• An intermediate solvent that is fully miscible with both ethanol and paraffin wax is needed to remove alcohol and other dehydrating solutions from tissues prior to embedding (usually in paraffin wax), and from finished slides prior to mounting.
• Clearing agents are also used after sectioning to remove paraffin wax after cutting on the microtome.
• Clearing (de-alcoholization) is the process whereby alcohol or a dehydrating agent is removed from the tissue and replaced with a substance that will dissolve the wax with which the tissue is to be impregnated (e.g. paraffin) or used as the medium on which the tissue is to be mounted (e.g. Canada balsam).
• Aside from removing alcohol, a clearing agent must also be miscible with Canada balsam and other resins that are used for mounting sections. This stage in the process is called “clearing” because many (but not all) clearing agents impart an optical clarity or transparency to the tissue due to their relatively high refractive index. This change in appearance is often used as an indication of the effectiveness or completeness of the clearing process.
• Because of the high refractive indices of most reagents used for de-alcoholization, tissues, particularly embryos and parasites, become transparent so that the internal structures become visible to the naked eye.
• Another important role of the clearing agent is to remove a substantial amount of fat from the tissue which otherwise presents a barrier to wax infiltration.
• The most commonly used clearing agent for this purpose is xylene. Glycerin and gum syrup are used when the tissue is to be cleared directly from water, as in a frozen section. No de-alcoholization is involved in this process.
• Characteristics of a Good Clearing Agent:

  • It should be miscible with alcohol to promote rapid removal of the dehydrating agent from the tissue
  • It should be miscible with, and easily removed by melted paraffin wax and/or by mounting medium to facilitate impregnation and mounting of sections
  • It should not produce excessive shrinkage, hardening or damage of tissue
  • It should not produce excessive shrinkage, hardening or damage of tissue
  • It should not evaporate quickly in a water bath
  • It make tissues transparent.
• Clearing fluids with a low boiling point are generally more readily replaced by melted paraffin, although chloroform which has a lower boiling point than xylene in fact takes longer than the latter to clear. Viscosity also affects the speed of penetration of the clearing agent. Prolonged exposure to most clearing agents causes the tissue to become brittle and therefore more difficult to cut.
• The choice of a clearing agent depends upon the following:

  • The type of tissues to be processed, and the type of processing to be undertaken
  • The processor system to be used
  • Intended processing conditions such as temperature, vacuum and pressure
  • Safety factors
  • Cost and convenience
  • Speedy removal of dehydrating agent
  • Ease of removal by molten paraffin wax
  • Minimal tissue damage
• Xylene (Xylol) - a colorless clearing agent that is most commonly used in histology laboratories. Clearing time is usually 1/2 to 1 hour. It is used for clearing, both for embedding and mounting procedures. It is generally suitable for most routine histologic processing schedules of less than 24 hours, and when the tissue block size is less than 5 mm. in thickness. Xylene is reasonably cost effective and works well for short-term clearing of small tissue blocks.
• Advantages of Xylene:

  • It is the most rapid clearing agent, suitable for urgent biopsies which it clears within 15-30 minutes.
  • It makes tissues transparent.
  • It is miscible with absolute alcohol and paraffin
  • It does not extract out aniline dyes.
  • For mounting procedures, it does not dissolve celloidin and can, therefore, be used for celloidin sections.
  • It evaporates quickly in paraffin oven and can, therefore, be readily replaced by wax during impregnation and embedding.
  • Cheap
• Disadvantages of Xylene:

  • It is highly inflammable and should be appropriately stored.
  • If used longer than 3 hours, it makes tissues excessively hard and brittle.
  • It causes considerable hardening and shrinkage of tissues; hence, is not suitable for nervous tissues and lymph nodes.
  • Xylene becomes milky when an incompletely dehydrated tissue is immersed in it
  • Xylene may irritate eyes, nose and respiratory tract. It can be absorbed through the skin and cause dermatitis. At high concentrations, it is toxic and narcotic
• Toluene is better at preserving tissue structure and is more tolerant of small amounts of water left behind in the tissues than xylene. However, toluene is more expensive than xylene and more toxic, so toluene is less commonly used. Toluene may be used as a substitute for xylene or benzene for clearing both during embedding and mounting processes. Time recommended for clearing is 1 -2 hours.
• Advantages of Toluene:

  • It is miscible with both absolute alcohol and paraffin.
  • It acts fairly rapidly and is recommended for routine purposes.
  • Tissues do not become excessively hard and brittle even if left in toluene for 24 hours.
  • Clears overnight
  • It is not carcinogenic
• Disadvantages of Toluene:

  • It is slower than xylene and benzene.
  • It tends to acidify in a partially filled vessel
  • Highly concentrated solutions will emit fumes that are toxic upon prolonged exposure.
  • It is more expensive.