Fresh tissue exam

• Methods of fresh tissue examination: teasing /dissociation, crushing/squash preparation, smear preparation, frozen section
• Smear preparation:

  • Streaking
  • Spreading
  • Pull apart
  • Touch preparation
• Frozen section:

  • Normally used when a rapid diagnosis of a tissue is required
  • Freezing of unfixed tissue is best for frozen section
  • Freezing of fixed tissue is used to localize hydrolytic enzymes and other antigens
• Applications of frozen section:

  • Rapid pathologic diagnosis during surgery
  • Enzyme histochemistry
  • Demonstration of soluble substances such as lipids and carbohydrates
  • Immunofluorescent and immunohistochemical staining
  • Some specialized silver stains, particularly in neuropathology
• Freeze drying without the use of any chemical fixative:

  • Quenching: rapid freezing (-160°C)
  • Sublimation: removal of water in the form of ice (-40°C vacuum)
• Freeze substitution:

  • Similar to freeze drying
  • Frozen tissue is submerged to Rossman's formula (1% acetone) and dehydrated using absolute alcohol
• Two methods of preparing frozen section:

  1. Cold knife procedure:

  • Optimum conditions for sectioning: knife (-40° to -60°C), tissue (5° to -10°C), environment (0° to -10°C)
  • Remedies for issues during sectioning
• 2. Cryostat procedure (cold microtome):

  • Optimum working temperature = -18°C to -20°C
  • Cryostat: a refrigerated cabinet in which a modified microtome is housed
  • Commonly used methods of freezing
• Staining methods:

  1. Polychrome methylene blue

  2. Alcoholic pinacyanol

  3. Thionine

  4. Hematoxylin and Eosin: progressive (no decolorizer)
• Tissue processing steps:

  1. Fixation

  2. Dehydration

  3. Clearing/dealcoholization

  4. Infiltration/impregnation

  5. Embedding

  6. Trimming

  7. Section/microtomy

  8. Staining

  9. Mounting

  10. Labelling
• Characteristics of a good fixative:

  • Cheap
  • Stable
  • Safe to handle
  • Prevent distortion
  • Inhibit bacterial decomposition
  • Produce minimum shrinkage
  • Rapid penetration of tissue
  • Harden tissue
  • Isotonic with minimal effect on tissue
  • Permit staining
• Mechanism of action of fixative:

  1. Additive fixation: becomes part of the tissue by formation of cross links or complexes

  2. Non-additive fixation: stabilizes tissue by removing bound water
• Factors involved in fixation:

  • pH
  • Temperature
  • Thickness
  • Osmolality
  • Concentration
  • Time duration
• Practical considerations for fixation:

  1. Speed

  2. Rate of penetration

  3. Volume

  4. Duration
• Cytoplasmic fixatives include: Helly’s fluid, Orth’s fluid, Regaud’s fluid, Flemming fluid without acetic acid, Formalin with post chroming
• Histological fixatives:

  • Lipid Fixation: Fixatives containing mercuric chloride and potassium dichromate in cryostat section
  • Carbohydrate Fixation: Alcoholic fixative for glycogen (Rossman’s fluid or cold absolute alcohol)
  • Protein Fixation: Neutral buffered formaldehyde
• • Glutaraldehyde: For electron microscopy
  • Karnovsky’s paraformaldehyde: For electron histochemistry and electron immunocytochemistry
  • Acrolein: Mixture with formaldehyde
  • Formol-calcium: For lipids in frozen sections
• Aldehyde fixatives:

  • Formaldehyde (Formalin): Gas produced by the oxidation of methyl alcohol. Concentrated solutions should not be neutralized to avoid explosion. Stock solution: 37-40%, Working solution: 10%. Formalin pigments include Paraformaldehyde and Acid formaldehyde hematin
  • 10% Formol Saline: Diluted in 10% NaCl CNS
  • 10% Neutral Buffered Formalin (NBF) or PO4 buffered formalin: Best general tissue fixative, best fixative for tissue containing iron granules with double phosphate buffer
  • Formol corrosive (formol sublimate): For routine post mortem tissues with HgCl2
• • Brasil’s alcoholic picroformol fixative: Excellent fixative for glycogen
• Metallic fixatives:

  • Mercuric chloride (BOSCHZZ):
  • B5 fixative: For BM biopsies
  • Ohlmacher’s
  • Schaudinn’s
  • Carnoy-Lebrun
  • Heidenhain Susa: For tumor biopsies, especially of the skin
  • Zenker formol (Helly’s solution): Fixative for pituitary gland, BM, and blood-containing organs
  • Chromate fixatives:
  • Regaud’s (mollers) (molliflex)
  • Orth’s fluid
  • Chromic acid
  • Potassium dichromate (K2CrO4)
  • Lead fixatives: Recommended for acid mucopolysaccharides
  • Picric acid fixative:
  • Bouin’s: Recommended for fixation of embryos and pituitary biopsies
• Glacial acetic acid:

  • Used in conjunction with other fixatives to form a compound solution
  • Solidifies at 17°C
  • Fixes and precipitates nucleoproteins, chromosomes, and chromatin material
• Alcohol fixatives:

  • Methyl alcohol: For fixing dry and wet smears
  • Ethanol: Simple fixative incorporated with compound fixatives, preserves but does not fix glycogen
  • Isopropyl alcohol: For fixing touch preparations
  • Carnoy’s fluid: For fixing chromosomes, lymph glands, and urgent biopsies
  • Alcoholic Formalin (Gendre’s fixative): To preserve sputum
  • Newcomer’s: For fixing mucopolysaccharides and nuclear proteins, gives better reaction in Fuelgen stain than Carnoys
• Osmium tetroxide:

  • Pale yellow powder that dissolves in water to form a strong oxidizing solution
  • Inhibits hematoxylin
  • Produces black precipitate crystals for lipids
  • Flemmings solution: Common chrome-osmium acetic acid fixative used for fat and nuclear structures
  • Flemming’s solution without acetic acid: Recommended for mitochondria
• Trichloroacetic acid:

  • Precipitates proteins
  • Counteracts shrinkage by other fixatives
  • Weak decalcifying agent
• Acetone:

  • Used at ice-cold temperatures for diffusible enzymes such as phosphatases and lipases
  • For fixing brain tissue (Rabies Diagnosis)
• Heat fixation:

  • Thermal coagulation of tissue proteins
  • For bacteriologic smears
  • Microwave: 45-55°C
  • Underheating leads to poor sectioning
  • Overheating (>65°C) leads to vacuolation and overstained cytoplasm
• Fixatives for enzyme histochemistry:

  • 4% formalin or formol saline, acetone, or formalin for cryostat sections
• Fixatives for electron microscopy:

  • Glutaraldehyde, PtCl3, PtCl3-formalin (Zamboni’s), AuCl, Osmium tetroxide
  • 10% NBF is acceptable but not recommended
• Fixative for electron histochemistry and electron immunocytochemistry:

  • Karnovsky’s paraformaldehyde glutaraldehyde
• Fixation terminologies:

  • Secondary fixation: Placing an already fixed tissue in a second fixative
  • Post-chromatization: Fixation whereby a primarily fixed tissue is placed in an aqueous solution of 2.5-3% potassium dichromate
  • Washing out: Removing excess fixative
• Factors affecting fixation of tissues:

  • Size and thickness of the tissue specimen
  • Presence of mucus, fat, blood
  • Cold temperature
• Principles and precautions in handling fixatives:

  • Autopsy/surgical materials should be fixed soon after death
  • Tissues must be properly labeled
  • Avoid refrigerating tissues at 0°C
  • Tissues should not be more than 5mm thick, except in lung edema
  • Purulent, exudates, or transudates should be kept for bacteriologic culture
  • Adequate amount of fixative (20:1 ratio)
  • Avoid tissue contamination
  • Wash tissues before staining
  • Solid organs should be injected with fixatives
  • Hollow organs should be packed with cotton soaked in fixative or opened
• • Air-filled lungs should be covered with gauze soaked in fixative
  • Human brain should undergo intravascular perfusion
  • Eyes should not be dissected before fixation
  • Muscle tissues should be stretched to avoid rigor
• Difficulties encountered because of improper fixation:

  • Failure to arrest early autolysis of cells due to delayed fixation
  • Removal of substances soluble in fixing agent due to the wrong choice of fixative
  • Presence of artifact pigments due to incomplete fixation
  • Soft and feather-like tissues due to incomplete fixation
  • Loss or inactivation of enzymes due to the wrong choice of fixative
  • Shrinkage and swelling of cells due to overfixation
  • Brittle and hard blocks due to prolonged fixation
• Pigment color removed by different substances:

  • Acid formaldehyde hematin: Removed by saturated picric acid, alcoholic KOH, Kardasewitsch method, Lillie’s method
  • Mercuric chloride pigment: Removed by alcoholic iodine
  • Chromate pigment: Removed by acid-alcohol
  • Osmium tetroxide pigment: Removed by cold H2O