TISSUE PROCESS OVERVIEW AND AUTOTECHNICON

• Histopathology


  The diagnosis and study of diseases of the tissues, and involves examining tissues and/or cells under a microscope
• Tissue processing


  The steps required to take human tissue from fixation to the state where it is completely infiltrated with a suitable histological wax and can be embedded ready for section cutting on the microtome
• Histotechnologist


  • Examining, fixing, and processing the tissue for proper sample preservation
  • Surrounding the tissue with paraffin wax in a process called embedding
  • Cutting a tissue sample with a microtome or cryostat
  • Mounting the tissue sample on a slide
  • Staining or dying the sample to emphasize abnormalities
• Specimen preparation


  1. Identification of specimen

  2. Materials used for gross examination

  3. Describing the specimen

  4. Sectioning
• Fixation


  • Preserve the morphologic and chemical integrity of the cell in as life-like manner as possible
  • Harden the tissue to protect it against subsequent processing steps
• Dehydration


  The process of removing intercellular and extracellular water from the tissue FOLLOWING FIXATION, and PRIOR to WAX IMPREGNATION
• Clearing


  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
• Infiltration


  The process of removing the clearing agent from the tissue and replaced by a medium that will completely fill all the tissue cavities, giving the specimen a firm consistency
• Embedding


  The tissues or the specimens are enclosed in a mass of the embedding medium using a mould
• Section-cutting


  The process of cutting tissue into thin slices
• Mounting


  Sections are mounted under cover slip to maintain high refractive index necessary for critical microscopy and to protect the section during storage
• Autotechnicon


  An automatic tissue processing machine that fixes, dehydrates, clears, and infiltrates tissues thereby decreasing the time and labor needed during the processing of tissues resulting in a more rapid diagnosis with lesser technicality
• Process involved in autotechnicon

  1. Fixation

  2. Dehydration

  3. Clearing

  4. Infiltration
• Factors that impact the duration of tissue processing and extent of infiltration

  • Tissue density and thickness
  • Agitation
  • Temperature
  • Vacuum and pressure
• Technical errors


  • Reagent carry-over
  • Power failure
  • Quality control
• Histopathology


  The study of the different diseases and pathologic processes that is found in the tissues
• Cytology


  The study of cells and what are the techniques used in order for you to assess the pathologic process that is happening in the cells and in the tissue
• Fresh tissue is usually examined where there is an immediate need for evaluation. A better and more effective means however, of studying tissues whether normal or abnormal is by examination of their sections and smears which have been permanently preserved. Stained for demonstration of specific structures and mounted on glass slides with cover slips for permanent keeping.
• Tissue Processing


  1. Fixation

  2. Dehydration

  3. Clearing

  4. Infiltration (Impregnation)

  5. Embedding

  6. Trimming

  7. Section-Cutting/Microtomy

  8. Staining

  9. Mounting

  10. Labelling
• Fixation


  The first and most critical step in histotechnology involving the fixing or otherwise known as the preserving of the fresh tissue for examination
• The quality of the section of the slide is only as good as the quality of the fixed tissue specimen. So if you properly fix your specimens, then you will get an optimum result for the pathologist to be read.
• Aim of fixation


  To preserve the morphology and the chemical integrity of the cell in as lifelike manner as possible
• The most common fixative used in the laboratory in Cebu Doctors' University Hospital is the "neutral buffered formaldehyde or otherwise known as NBF neutral buffered formalin.
• Dehydration


  The process of removing the intercellular and extracellular water from the tissue following fixation and before wax impregnation
• The most common dehydrating agent used in the histopath lab is "ethyl alcohol" so we start from a smaller percentage or concentration going to a higher or increasing strength of alcohol concentration.
• Whatever the dehydrating agent that is used, the amount in each stage should not be less than 10 times the volume of the tissue.
• Clearing


  The dehydrating agent is removed from the tissue and replaced with a substance that will dissolve the wax with which the tissue will be impregnated, giving the tissue a "translucent appearance"
• Clearing agents


  • Chloroform
  • Toluene, xylene, Benzene
  • Carbon Tetrachloride
  • Cedar wood oil
  • Methyl Benzoate, Methyl Salicylate
  • Citrus Fruit Oil
• The most common clearing agent used in the laboratories is "xylene".
• Infiltration (Impregnation)


  The process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will completely fill all the tissue cavities, thereby giving a firm consistency to the specimen, and allowing easier handling and cutting of suitably thin sections without any damage or distortion to the tissue and its cellular components
• Embedding


  The process by which the impregnated tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify
• Orientation


  The process by which a tissue is arranged in precise position in the mold during embedding on the microtome before cutting and on the slide before staining
• Trimming


  The process of removing excess wax after embedding
• Section-Cutting/Microtomy


  The process by which a processed tissue, most commonly a paraffin embedded tissue, is trimmed and cut into uniformly thin slices or "sections" to facilitate studies under the microscope
• Staining


  The process of applying dyes on the section to see and study the architectural pattern of the tissue and physical characteristics of the cells
• The most common staining technique used in the histopath lab is "H and E staining".
• Mounting


  The process of placing a cover slip over the top of the stained tissue section
• The most common mounting media used in the histopath lab in CDU is Eukitt.
• Labelling


  One of the most important steps, where you must properly label the tissue section from fixation to labeling
• Any problems within the process will mess it up. Label your slides with a pencil to avoid mixing with others or losing it.