Tissue Processing

Standard tissue processing may be carried out on any number of open and closed tissue processors, although closed processors are preferred due to safety concerns, both for the tissues and laboratory personnel.  Closed system processors are “smart’ enough to prevent tissues from drying out in the event of a power failure, and the chemical fumes are kept inside the processor; released through filters and/or vented to the outside of the laboratory space.

Another tissue processing option is the use of microwave assisted processors that use conventional heat and microwaves to adjust and maintain temperature control during processing.  Specimens are dehydrated through ethanol and isopropanol.  Then, after vacuum vaporization, specimens are infiltrated with molten paraffin.  The specimens are then ready for embedding.  There are also ancillary units that will perform automated embedding of the tissues, if the proper cassettes are used.

A major advantage of microwave assisted tissue processors is the rapidity of processing of biopsy specimens.  Small tissue biopsies of skin, prostate and gastrointestinal tissue can be processed into paraffin in approximately one hour.  This is extremely valuable for cases requiring “rush” status.  Another advantage is that, since no xylene is used, the tissues are generally much softer in the paraffin block, and therefore much easier to cut during microtomy, resulting in fewer cutting artefacts.

Processing procedures using microwave assisted tissue processors must be clearly and accurately defined, with much attention during the validation process.  The fixation and dehydration steps must be complete to ensure proper infiltration with molten paraffin.  Like routine tissue processors, the basic stages of tissue processing must accomplish:

  1. Fixation of tissue to stabilize proteins and harden the tissue
  2. Dehydration of tissue to remove all unbound water
  3. Clearing of tissues to remove the dehydrant
  4. Infiltration of tissue with molten paraffin, to ensure the embedding process is successful

 

There are many factors involved in tissue processing, which provide many opportunities for things to go awry.  Carry over of fixative into the processing alcohol can inhibit subsequent dehydration.  If the absolute alcohol stations prior to the clearing stations contain water, this will result in incomplete dehydration as well.  When water is left in the specimen, it cannot be removed by the clearant, and becomes trapped within the tissue during paraffin infiltration.  The resulting paraffin blocks will be soft and difficult to cut during microtomy.

Conversely, tissue can become “over dehydrated” if the processing times in alcohol are too long.  Tissues contain an amount of molecular “bound water’ within the nuclei and some other tissue elements.  If this water is removed during extended dehydration, the resulting paraffin blocks may be dry, scratchy, and hard to cut during microtomy.  Soaking in cold ice water, once the block is faced off, may sometimes be used as a remedy.  This is common in laboratories that use only one tissue processor to process all of their tissues, regardless of size and type.  In this case, smaller tissues (i.e. biopsies) may become dry and brittle for cutting.

The next blog will discuss specific tissue processing artefacts that are observed in the microscope slide.  Now that you have a background in the chemistry and rationale of tissue processing, you will be able to understand how you can troubleshoot and remedy these all too common processing artefacts.

 

References:

Chapman, C.M. (2017). The Histology Handbook: Amazon CreateSpace Independent Publishing Platform

Chapman, C.M. and Dimenstein, I.B. (2016). Dermatopathology Laboratory Techniques. Amazon CreateSpace Independent Publishing Platform

Chapman, C.M. (2018). Troubleshooting in the Histology Laboratory.  Submitted to J Histotechnol

 

Questions and comments may be sent to articles@rankinbiomed.com

 

Clifford M Chapman
Senior Consultant
Medi-Sci Consultants