lec 2

• Tissue Processing (Fixation and Dehydration)
• Fixation


  First and most critical step in tissue preservation for pathologic diagnosis
• Fixation


  • Stops metabolic processes
  • Allows fixative to penetrate deeply into the tissue
  • Hardens the tissue for easy cutting and cleaning
• Primary aim of fixation

  Preserve the morphologic and chemical integrity of the cell in a life-like manner
• Secondary aim of fixation

  Harden and protect the tissue against any further processing and handling
• Functions of Fixation


  1. Preserve the tissue

  2. Prevent breakdown of cellular elements

  3. Coagulate/precipitate cytoplasmic substances
• Practical considerations to optimize fixation

  • Speed
  • Volume
  • Penetration
  • Duration of Fixation
  • Osmolality
  • Concentration
  • Temperature
  • Thickness
• Speed


  Specimen should be placed in fixative as soon as possible, less than an hour after surgery
• Volume


  Must be 20 times the size of the tissue
• Penetration


  Formalin diffuses into the tissue approximately 1 mm/hr, sufficient time must be allowed for proper penetration
• Duration of Fixation


  Primary fixation & buffered formalin: 2-6 hours
• Osmolality


  • Light microscopy: slightly hypertonic (between 400-450 mOsm)
  • Electron microscopy: more or less isotonic (340 mOsm)
  • 10% Neutral Buffered Formalin (NBF): 1500 mOsm
• Concentration


  • Formaldehyde = 10% solution
  • Glutaraldehyde = 3% solution and 0.1% solution
• Temperature


  • Fixation is done generally at room temperature (20 to 22 C)
  • Routine Automated: 40 C
  • Electron Microscopy and some histochemistry: 0 to 4 C
  • Formalin heated at 60 C: rapid fixation for very urgent biopsies
  • Formalin at 100 C: Diagnosis of TB
  • DNA: 65 C
  • RNA: 45 C
  • Microwave: 65C
• Thickness


  • If tissue sample is small: 1-2 mm for electron microscopy, 2 cm2 for light microscopy
  • Large solid tissue should be opened or sliced thinly to improve penetration of fixatives
• Dehydration


  Definition and Purpose
• Overview of Tissue Processing: Fixation, Dehydration, Clearing, Infiltration, Embedding, Trimming, Section Cutting, Staining, Mounting, Labeling
• Staff involved during tissue collection: Operating staff, Medical technologist
• Preservation of Tissue Sample is done the moment the tissue is removed from the patient's body and is to be placed in a fixative
• Tissues should be handled carefully, as it will be examined accurately by the Pathologist for proper diagnosis
• In the histopathology section, it is important that the principles and practice of fixation is done accurately
• The consequences of poor fixation is thoroughly understood by all the staff involved during the collection and processing of tissue samples
• Upon removal of the tissue specimen in the operating room, the tissue specimen must immediately be placed in a fixative
• To ensure that a solution is added to a specimen, we need to fix the tissue correctly so that the histotech can prepare good quality slides and for the pathologist to correctly and accurately diagnose the patient to be treated appropriately
• Upon the arrival of the specimen in the laboratory it is immediately placed in a liquid fixative agent, either formaldehyde solution or 10 % buffered neutral formalin
• Placing a fixative in a tissue will slowly penetrate in the tissue causing chemical and physical changes that will harden and preserve the tissue and protect it against any subsequent processing steps
• Initial fixation must be at least 2-6 hours before gross examination to ensure proper fixative penetration, making it easier for the pathologist to trim and cut the tissue
• After initial fixation or preliminary fixation, follows gross examination by the pathologist
• After the gross examination, the tissue is placed in a labeled cassette and submerged deeply into a 10% buffered neutral formalin fixative, which serves as a holding fixative that keeps the cassette ready for tissue processing
• High concentration


  May adversely affect the tissue and produce artifacts
• Fixation temperature


  • Room temperature
  • Routine Manual: 20 to 22 C
  • Routine Automated: 40 C
  • Electron Microscopy and some histochemistry: 0 to 4 C
  • Formalin heated at 60 C: rapid fixation for very urgent biopsies
  • Formalin at 100 C: Diagnosis of TB
  • DNA: 65 C
  • RNA: 45 C
  • Microwave: 65C
• Nucleic acids do not react with fixatives in room temperature
• Tissue sample size for electron microscopy

  1. 2 mm
• Tissue sample size for light microscopy

  2 cm
• Tissue sample measurements should not be compromised in order to obtain full penetration and satisfactory fixation
• Large solid tissue, such as a uterus, should be opened or sliced thinly to improve the penetration of fixatives
• Fecal matter and stomach contents inhibit the penetration of fixative and can cause damage to the tissue during sectioning
• Most tissues can be cut and trimmed without prior fixation, except for the brain
• Brain tissue fixation


  1. Suspended whole in 10% buffered formalin for about 2-3 weeks

  2. Fixation hardens the consistency of the brain and makes it easier to cut

  3. Brain tissue must be fixed before grossing/sectioning

  4. Incomplete fixation of the brain tissue will lead to improper and incomplete clearing and impregnation later on, which hinders normal sectioning and staining of the specimen
• Characteristics of an ideal dehydrating solution

  • Cheap and Economical
  • Stable and safe to handle
  • Fast acting, permits rapid and even penetration
  • Inhibits bacterial decomposition and autolysis
  • Must harden the tissue
  • At least isotonic
  • Must render tissues insensitive to subsequent processing
  • Must be compatible with many staining procedures