CEP Med - Tissue Proccessing

• What is Tissue processing?

  The various steps required to take the 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
• What does Tissue processing do?

  • Enables the pathologist to diagnose the presence or absence of disease
  • Produces a tissue section of good quality that allows for adequate interpretation of microscopic cellular changes
• Tissue processing


  • Bone tissue
  • Skeletal muscle
• Steps in Tissue processing:

  1. Fixation

  2. Decalcification (optional)

  3. Dehydration

  4. Clearing

  5. Impregnation (infiltration)

  6. Embedding

  7. Trimming

  8. Section Cutting (microtomy)

  9. Staining

  10. Mounting

  11. Labeling
• What is Fixation?

  • The first and most critical step in histotechnology
  • A process that preserves tissues from decay, thereby preventing autolysis or putrefaction
• What does Fixation do?

  • Preserves the tissue
  • Prevents breakdown of cellular elements
  • Coagulates or precipitates protoplasmic substances
• What is Decalcification?

  Removal of calcium ions from a bone or calcified tissue to make flexible and easier to cut
• What is Dehydration?

  The process of removing water from the tissue following fixation and prior to wax impregnation
• Dehydration routine

  1. 70% ethanol 15 min

  2. 90% ethanol 15 min

  3. 100% ethanol 15 min

  4. 100% ethanol 15 min

  5. 100% ethanol 30 min

  6. 100% ethanol 45 min
• Clearing


  Where 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 or used as the medium on which the tissue is to be mounted
• Clearing


  • Imparts an optical clarity or transparency to the tissue
  • Removes a substantial amount of fat from tissue which otherwise presents a barrier to wax infiltration
• Clearing


  1. Xylene 20 min

  2. Xylene 20 min

  3. Xylene 45 min
• 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 and give firm consistency to the specimen
• Embedding


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


  • Paraffin wax
  • Celloidin (Collodion)
  • Gelatin
  • Plastic
• Paraffin wax


  • Simplest, most common, and best embedding medium used for routine tissue processing
  • Solid at RT, but melts at 65-70C (usually at 60C)
  • Maximum temperature is 2-5C above the melting point
• Impregnation and embedding


  1. Paraffin wax 30 min

  2. Paraffin wax 30 min

  3. Paraffin wax 45 min
• Section-cutting (microtomy)


  Where processed tissue is trimmed and cut into uniformly thin slices or "Sections" to facilitate studies under the microscope
• Section-cutting (microtomy)


  The spring-balanced teeth or pawl is brought into contact with, and turns a ratchet feed wheel connected to a micrometer screw, which is in turn rotated, moving the tissue block at a predetermined distance towards the knife cutting sections at uniform thickness
• Common problems in section-cutting

  • Sections fail to form ribbons (surfaces and edges of the block are not parallel → re-trim the block)
  • Hole formed in the section (bubble or dirt from embedding medium → re-embed with freshly filtered wax)
  • Tissue shrinks away from wax when trimmed (insufficient dehydration → repeat the whole procedure)
• Staining


  Process whereby tissue components are made visible in microscopic sections by direct interaction with a dye or staining solution
• Methods of staining


  • Direct staining
  • Indirect staining
  • Progressive staining
  • Regressive staining
  • Differential staining
  • Metachromatic staining
  • Vital staining
  • Intravital staining
  • Supravital staining
• Direct staining


  Process of giving color to the sections by using aqueous or alcoholic dye solutions
• Indirect staining


  Process whereby the action of the dye is intensified by adding another agent or a mordant which serves as a link or bridge between the tissue and the dye to make the reaction possible
• Indirect staining - accentuator


  Does not participate in the staining reaction but merely accelerates the reaction
• Progressive staining


  Process whereby tissue elements are stained in a definite sequence, and the staining solution is applied for specific periods of time or until the desired intensity of coloring of the different tissue elements is attained
• Regressive staining


  Tissue is first overstained and the excess stain is removed or decolorized from unwanted parts of the tissue, until the desired intensity of color is obtained
• Differentiation (decolorization)


  The selective removal of excess stain from the tissue during regressive staining in order that a specific substance may be stained distinctly from the surrounding tissues
• Hematoxylin and Eosin (H&E) staining


  • Cornerstone of tissue-based diagnosis
  • Hematoxylin dye (basic dye) stains cell nuclei due to nuclear DNA (blue or purple)
  • Eosin dye (acidic dye) stains cytoplasm (pink or red)
• Differential staining


  Uses more than one chemical stain to better differentiate various microorganisms or structures/cellular components of a single organism
• Metachromatic staining


  Entails the use of specific dyes which differentiate particular substances by staining them with a color that is different from that of the stain itself (metachromasia)
• Vital staining


  The selective staining of living cell constituents, demonstrating cytoplasmic structures by phagocytosis of the dye particle, or by staining of pre-existing cellular components
• Intravital staining


  The selective staining of living cell constituents by injecting the dye into any part of the animal body (either intravenous, intraperitoneal, or subcutaneous), producing specific coloration of certain cells
• Supravital staining


  Method of staining to examine living cells that have been removed from an organism
• Mounting


  The process where the paraffinized ribbons are taken from the microtome knife and transferred to the warm water bath (5-10C below melting point of the embedding wax)
• Misidentification (one of the most serious issues) - due to "shedding" of friable small tissue fragments in the water "Floaters" tiny unrelated tissue causing misinterpretation by the pathologist