8 Frozen Section & Rapid Tissue Processing

• Frozen sections are especially recommended when lipids and nervous tissue elements are to be demonstrated

  • Usually done on muscles and nerve biopsies, as well as surgically removed tumors
  • Necessary in rapid diagnosis process
• Immediate diagnosis is accomplished through frozen section biopsy.

  • Especially in intraoperative pathology to help the surgeon on his next plan of action
• Frozen section process:

  • Fresh tissue is frozen on a microtome with CO2 or on a cryostat, a chamber kept at an atmospheric temperature of -10 to -20 °C.
  • Very thin slices are made around 10-15 µm in thickness.
  • Frozen sections are transferred to a slide.
  • adhesives are not required.
  • Microscopic examination.
• Frozen section has a disadvantage of having poor quality of the final slide

  • TO REMEDY: Right after we do frozen section, we then place the specimen in a fixative and do permanent sections of that particular tissue.
• It is very important and required that all frozen sections have permanent section

  • this will be able to help the pathologist check the diagnosis of frozen section in relation to permanent section.
  • we are able to have more tissue samples and at the same time more areas that we are able to check and compare in the frozen section.
  • we can see much more in sample from permanent section than in frozen section
• The tissue for freezing should be fresh and should be frozen as quickly as possible in order to avoid or prevent autolysis
• Slow freezing can cause distortion of tissue due to ice crystal artifacts.
• Liquid nitrogen is generally used in histochemistry and during intra-operative procedures; most rapid among the commonly available freezing agents.
• Liquid nitrogen method

  A) Crystals

  B) Freezing artifacts

  C) vapor phase

  
• Isopentane is liquid at room temperature; it is an excellent method for freezing muscle tissue.
• In isopentane method,

  • Pyrex glass beaker containing isopentane is suspended in a flask of liquid nitrogen until half-liquid & half-solid.
  • If half-liquid & half-solid phase is achieved, beaker is removed from liquid nitrogen when small crystals start forming.
  • Tissue to be frozen is dropped
• Carbon dioxide uses convention freezing microtome with gas supply of carbon dioxide gas from a carbon dioxide cylinder

  • In SPMC, they are using a conventional freezing microtome, which is used when the cryostat is under repair and there are frozen section biopsies
• Aerosol sprays become increasingly popular method; adequate for freezing small pieces of tissue except muscles.

  • It rapidly freezes blocks of any type of tissue.
  • Quick-freeze spray cans of fluorinated hydrocarbons rapidly freezes blocks of any type of tissue [e.g., Cryokwik]
• In Cold knife procedure, tissue blocks can be frozen by adapting a conventional freezing microtome gas supply of carbon dioxide gas from a carbon dioxide cylinder or by using a cryostat.

  • Almost any microtome can be utilized for the purpose provided means are made available for freezing and maintaining the specimen and the knife at low temperature, usually by utilizing the carbon dioxide technique.
• In cold knife procedure,

  • The success of this procedure depends upon the ambient temperature and humidity = prone to errors
  • Sections thinner than 6 μm generally cannot be obtained even from tissues that section well and with ideal conditions for sectioning.
• When using cold knife in a controlled cold environment, optimum condition for sectioning shall be provided.

  • Knife: -40 to -60 °C
  • Tissue: -5 to -10 °C
  • Environment: 0 to -10 °C
• Cold knife procedure:

  • A piece of filter paper soaked in gum syrup is placed on microtome stage.
  • Short bursts of CO2 are applied
  • Selected tissue block 3-5 mm thick is oriented on stage.
  • Apply few drops of gum syrup and frozen solid with several intermitted bursts of CO2.
  • Tissue is lifted up to knife manually and trimmed until surface is flat.
  • Surface is warmed with finger until hard frozen tissue starts to thaw.
  • Sections do not form ribbons but stick to knife blade
  • Sections are transferred to a dish of distilled water to separate and picked up individually for mounting and staining.
• DewLine is the point where sections may then be cut at 10um thickness
• In cold knife procedure, sections stick to the knife blade, the section should be removed using a camel hair-brush or a finger moistened with water
• The water dish is usually placed on a dark or black background in order to see the sections, which are usually colorless or very light in color
• Cryostat procedure or Cold microtome makes use of the cryostat, an apparatus used in fresh tissue microtomy.

  • Optimum working temp of cryostat should be 18 to -20 °C
  • Majority of sections can be cut in isothermic condition.
  • The tissue for freezing should be fresh, and freezing should be done as quickly as possible.
  • Slow freezing can cause tissue distortion due to ice crystals artifacts.
• Fresh frozen tissues requires that the tissue be maintained in the frozen solid state during cutting of section thereby supporting and protecting the tissue for any damage and distortion by the knife during the process of cutting.
• Freezing shelf is the area of cryostat where tissue samples are frozen, or where frozen are stored prior to sectioning.

  • WORKING TEMP: -10 °C
• Heat extractor of the cryostat is used to maintain low temp of the area.
• Anti-roll guides of the cryostat helps to prevent the rolling or curling of sections being prepared; consists of glass plates supported in a metal or aluminum frame.
• Each turn of the Cryostat hand wheel advances the tissue block from the precise increment of section thickness setting; w/ locking mechanism.
• A) Heat extractor

  B) Freezing shelf

  
• A) Knife holder

  B) Anti roll guides

  
• A) Cryostat hand wheel

  B) Locking mechanism

  
• Synthetic Water-soluble Glycols & Resins are generally used as mounting media for tissue block that need to be sectioned on a cryostat

  • It provides different plastic dispensers in different temperature ranges
  • -5 to -15 °C → for brain, lymph nodes, liver, spleen, uterine, curetting, and soft cellular tumors.
  • -15 to -25 °C → for non-fatty breast tissue, ovary, prostate, tongue, and GI tract.
  • -35 °C → for fatty breast and omental tissue.
• In mounting of frozen tissue section,

  • Cryostat is usually set at -18 to -20 °C and tissue block should be 2-4 mm thick to maximize size of knife hitting the metal tissue block holder.
  • Frozen tissue is mounted on microtome.
  • Both microtome knife and tissue block are left in cryostat for 15 mins at -20 °C.
  • Sections between 5-10 μm are slowly cut and steadily removed, attached directly to slides of cover-glasses, air dried and fixed
• Cryostat sections basically provide the simplest, quickest, and least labor-intensive method for producing frozen sections.

  • They are actively routinely used for intraoperative and rapid diagnosis of surgical specimen.
  • It should be noted that cryostat cut only individual sections, this is to compare it with your paraffin blocks which is able to form ribbons during cutting or sectioning.
• Cryostats sections of fresh unfixed tissue usually attach easily to the slide even without adhesives and will preserve enzymes and other substances that may be studied by histochemical techniques
• Cryostat is also recommended for any technique requiring cold sectioning of fixed materials such as fats, lipids, and some special methods for the nervous system.
• Formalin-fixed sections may not adhere to slides

  • Clean slides should be coated with albumin or chrome-glycerin jelly to adhere.
  • Immersed tissue block should be immersed in boiling 10% buffered formalin for 1-2 minutes before freezing and sectioning
• Special fixatives (10% formol calcium at 4°C) may be used in histochemistry for lipid demonstrations.
• Fixed tissue stored in alcohol should be washed in water for 12-24hrs before sectioning since it inhibits freezing.
• In Histochemical evaluation involving enzyme studies, tissues need to be chemically active, and chemical constituents should be present, unaltered nor displaced.
• Frozen section is deemed to be the ideal and preferred means of preserving tissue in order to avoid complete or partial loss of enzymes.
• If enzyme will be evaluated or studies, the best and the most ideal means is to have it preserved in frozen section