HISTOPATHOLOGICAL TECHNIQUES

• Gross examination aka Grossing is when pathologist describes and obtains measurements of the specimen.

  • small pieces of tissues are cut for tissue processing.
  • the medical technologist assists in this process
• Fresh tissues have the advantage of being examined in the living state, allowing protoplasmic activities (motion, mitosis, phagocytosis, pinocytosis) to be observed.
• Teasing or Dissociation is the process where tissue specimen is immersed in a watch glass containing isotonic salt solution either stained or unstained.

  • viewed in Phase Contrast or Bright Field Microscope
• Squash preparation or Crushing is the process where tissues less or equal to 1mm in diameter are pressed between 2 slides or between a slide and a cover glass.

  • Vital stain is applied between the slides.
  • viewed in Phase Contrast or Bright Field microscope
• Smear Preparation is the process of examining sections or sediments where cellular materials are spread lightly over a slide by means of a wire loop, applicator stick or another slide.

  • includes streaking, spreading, pull-apart, and touch preparation.
  • this technique is especially useful in cytological examinations especially for cancer diagnosis.
• Streaking is a smear preparation process where tissues are rapidly and gently applied in a direct or zigzag line throughout the slide.
• Spreading is a smear preparation process where tissues are gently spread material into a moderately thick film; recommended for fresh sputum, bronchial aspirates, and thick mucoid secretions.
• Pull-Apart is a smear preparation process where tissues a drop or sediment is placed on a slide, faced into another slide, and is pulled apart in a single, uninterrupted motion.

  • This is recommended for thick secretions including serous fluids, concentrated sputum, enzymatic lavage samples from GI tract and blood smears.
• Touch preparation or Impression smear is a smear preparation process where surface of a freshly cut piece of tissue is brought into contact and pressed on the surface of a clean glass slide.

  • The cells are examined without destroying their actual intercellular relationship.
• Frozen section is normally used when a rapid diagnosis of tissues is required
• Frozen section uses a Cryostat, a cold chamber kept at an atmospheric temperature of -10-20°C which contains a modified Rotary microtome.
• After gross examination, tissues are placed in plastic cassettes and are subjected to subsequent tissue processing.
• Fixation is one of the most critical step in histotechnology; a process that preserves tissues from decay, thereby preventing autolysis and putrefaction.

  • the process should be carried out as soon as possible after removal of tissue from the body
• Main factors involved in fixation

  • Hydrogen Ion Concentration → pH 6-8
  • Temperature → for routine surgical specimens, use room temperature.
  • Thickness of the section (block):
  • Electron microscopy: 1-2 mm2
  • Light microscopy: 2 cm2
  • Osmolality
  • Hypertonic – causes cell shrinkage.
  • Isotonic & Hypotonic – causes cell swelling and poor fixation.
• Main factors involved in fixation

  • Concentration of solutions
  • Formaldehyde – most commonly used as 10% solution.
  • Glutaraldehyde – used as 3% solution.
  • Duration of fixation
  • Prolonged fixation = may cause cell shrinkage and hardening of tissues.
  • Incomplete fixation = softening of tissues, very hard to cut during section cutting.
• In fixation,

  • Large tissues like uterus should be opened/sliced thinly.
  • Brain is suspended whole for 2-3 weeks.
• Slightly hypertonic solutions with 400-450 mOsm for fixation = best results
• Use of Low concentration of glutaraldehyde (0.25%) in fixation is ideal for immunoelectron microscopy
• The ideal fixative to tissue ratio is 20:1
• Microanatomical fixatives is used for general microscopic study of tissue structures.
• Cytological fixatives is used for specific parts and particular microscopic elements of the cell itself.
• Nuclear fixatives preserves nuclear structures of cell; usually contains glacial acetic acid (pH 4.6 or less) which fixes nucleoprotein.
• Cytoplasmic fixatives is useful for preservation of cytoplasmic details or structures (pH greater than 4.6); must never contain glacial acetic acid bc it destroys mitochondria and Golgi bodies.
• Histochemical fixatives preserves chemical constituents of the cell.
• Washing out is the process of removing excess fixative from the tissue after fixation in order to improve staining and remove artefacts from the tissues
• Decalcification is the process of removing calcium ions from a bone or calcified tissue through a histological process that makes them flexible and easier to cut.
• Principle of Decalcification: Strong mineral acids or chelating agents form soluble calcium salts in an ion exchange process that moves calcium into decalcifying solution.
• Poorly fixed specimens becomes macerated during decalcification and stain poorly afterwards.
• Recommended ratio of fluid to tissue volume in decalcification is 20:1
• Ideal time required for complete decalcification is 24-48 hours but also varies with tissues and the type of agent used.
• Optimal temperature in decalcification is room temperature.
• If chemical method of decalcification is to be done, the decalcifying agent should be prepared with distilled water since false positive readings may be produced by calcium ions present in tap water.
• Tissue softener is used for unduly hard tissues that may damage microtome knives.
• Dehydration is the process of removing intercellular and extracellular water from the tissue after fixation and prior to impregnation

  • it is necessary to remove the fixative and water from the tissue and replace them with dehydrating fluid in preparation for impregnation.
• General rule in dehydration:

  • The amount in each stage should not be less than 10x the tissue volume
• Dehydrating fluids are generally used in increasing strengths; it provides little disruption to tissue due to diffusion currents
• Ethanol is used for routine dehydration and considered as the best dehydrating agent.
• Methyl Alcohol is a dehydrating agent employed for blood and tissue films.
• Acetone is both a fixative and dehydrating agent.
• Dioxane and Tetrahydrofuran is both a dehydrating and clearing agent.