10 Tissue Processing

• Frozen section is for rapid diagnosis, to optimally process tissues for special studies for diagnosis, treatment, or research, and to confirm that lesional tissue is present for diagnosis on permanent sections
• Optimal turn-around time for rapid frozen section is < or = 15 mins.
• Cryostat for rapid frozen section is -17 °C
• In Additive fixation, chemical constituent of fixative is taken in & becomes part of the tissue by cross-links or molecular complexes = stable protein
• Non-additive fixation removes bound water by attaching to H bonds of certain groups within the protein molecule → new cross links are established
• Microwave technique uses physical agent like vacuum, oven (heat) and agitation to increase the movement of molecules and accelerate fixation.

  • It accelerates staining, decalcification, immuno-histochemistry, and electron microscopy
• Oscillation frequency of microwave technique is 2450 mHz
• Microwave technique

  
• Fixative is cheap, stable, safe to handle, kills quickly, minimum tissue shrinkage, rapid and even penetration, hardens tissues for easier cutting, and isotonic
• Simple fixatives according to composition:

  • Aldehydes
  • Metallic fixatives
• Cytological fixatives according to action:

  • Nuclear
  • Cytoplasmic
• Formaldehyde (HCHO) is a simple fixative that is cheap, readily available, easy to prepare, stable, compatible w/ stains, penetrates tissues well, preserves fat, mucin, glycogen, for tissue photography.
• When methanol is oxidized, formaldehyde is formed
• Irritating fumes, prolonged fixation in formaldehyde = bleach tissues
• Glutaraldehyde is a simple fixative used for light microscopy and EM.

  
• Mercuric Chloride is the most common metallic fixative; 5-7%

  • for tissue photography, recommended for renal tissues, fibrin, CT, muscles.

  
• Zenker’s (HgCl2 + Glacial HAc) is a mercuric chloride used for liver, spleen, CT fibers, nuclei

  • has poor penetration, wash thoroughly in running H20.
• Zenker-Formol or Helly’s (HgCl2 , K2Cr2O7) is a mercuric chloride for pituitary, BM, spleen, liver

  • brown pigment is produced → remove by picric/NaOH
• Heidenhain’s Susa (HgCl2, NaCl, TCA) is a mercuric chloride for skin biopsies

  • place in high grade ROH.
• Chromic acid preserves CHO.
• Potassium Dichromate is a chromate fixative that preserves lipids, mitochondria
• Regaud’s or Moller’s (3% K2Cr2O7) is a chromate fixative for chromatin, mitochondria, Golgi, RBC, colloid, mitotic figures

  • slow, not for fats.
• Orth’s (2.5% K2Cr2O7) is a chromate fixative for Rickettsia, bacteria, myelin.
• Lead Fixatives are simple fixative that is for acid mucopolysaccharidosis (MPS); fixes connective tissue mucin.

  • forms insoluble lead carbonate → remove by filtering or adding Hac.
• Picric Acid fixatives is a simple fixative that yellow in color; highly explosive when dry

  • remove yellow color by 70% ethanol followed by 5% sodium thiosulfate & running water.
• Bouin’s (picric, HCHO, glacial) is a picric acid fixative used for embryos, glycogen and does not need washing out

  • has poor penetration, not good for kidneys, mitochondria, and hemolyzes RBC.
• Brasil’s alcoholic picroformol (w/ TCA) is a picric acid fixative good for glycogen

  • better & less messy than Bouin’s.
• Glacial Acetic Acid solidifies at 17°C; for nucleoproteins, chromosomes.

  • contraindicated in cytoplasmic fixatives → destroys mitochondria & Golgi.
• Polarization is used for glycogen
• Methanol is an alcohol fixative used in bone marrow and blood smears; slow
• Ethanol is an alcohol fixative and a strong reducing agent
• Carnoy’s-absolute (ROH, CHCl3, glacial HAc) is an alcohol fixative that is most rapid; RBC hemolysis.
• Alcoholic Formalin or Gendre’s is an alcohol fixative for sputum
• Newcomer’s is an alcohol fixative that is a mixture of isopropyl ROH, propionic acid, petroleum ether, acetone, dioxane → for MPS.
• Osmium Tetroxide or Osmic Acid is a simple fixative that fixes fats; for electron microscopy.

  • expensive, poor penetration, reduced w/ sunlight → black deposit; stored in dark bottle.
  • inhibits hematoxylin
  • acid vapor → causes conjunctivitis, osmic oxide in cornea = blindness.
  • extremely volatile
• Trichloroacetic Acid is a weak decalcifying agent; has poor penetration.
• Acetone is used at ice cold temp (-5-4°C); fixes brain → for rabies.

  • dissolves fat, evaporates rapidly, preserves glycogen poorly.
• 10% Formol Saline is a microanatomical fixative that penetrates and fixes tissues well, minimum shrinkage & distortion, does not overharden tissues; slow (>24 h).
• 10% Neutral Buffered Formalin is a microanatomical fixative used in preservation and storage of surgical, postmortem and research specimens

  • Na dihydrogen PO4, Disodium H PO4
  • best fixative for iron pigments, elastic fibers
  • longer to prepare → time consuming, inert towards lipids
• Formol Sublimate or formol corrosive is a microanatomical fixative that causes minimum shrinkage and hardening; no need for wash out from fixative to ROH.

  • formol mercuric chloride
  • slow
  • forms mercuric chloride deposits