Histopath - M6 - Special tissue processing PDF

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SPECIAL TISSUE PROCESSING TECHNIQUES Frozen section      ➢ Is a pathological laboratory technique used for rapid microscopic analysis / diagnosis of a specimen / disease      ➢ Usually used with oncologic surgery      ➢ Rapid diagnosis can guide intra-operative patient management      ➢ Recommended when lipids and nervous tissue elements are to be demonstrated      ➢ Used for:               • Rapid pathologic diagnosis during surgery               • Diagnostic and research enzyme histochemistry               • Diagnostic and research demonstration of soluble substances such as lipids and carbohydrates               • Immunoflurescent and immunocytochemical staining               • Some specialized silver stains,particularly in neuropathology     ➢ Methods of preparing frozen sections: • Cold knife procedure ▪ Sections do not form ribbons but rather stick to the knife blade  ▪ Sections are removed with a camel brush or finger moistened with water  ▪ Success of the procedure depends upon o Ambient temperature (facility of cutting and quality of sections are improved if a cold room is utilized) o Humidity (it is hard to cut sections in a hot or humid surroundings)             • Cryostat procedure (cold microtome) ▪ Makes use of the cryostat  o Consist of an insulated microtome housed in an electrically driven refrigerated chamber maintained at teperatures near -20°C,where microtome, knife, specimen and atmosphere are kept at the same temperature  o Optimum working temperature is -18°C to -20°C       ▪ Sections are cut in isothermic conditions       ▪ Tissue for freezing must be fresh o Freezing should be done as quickly as possible  - Slow freezing can cause distortion of tissue due to ice crystals artifacts     ▪ Use synthetic water-soluble glycols and resins as mounting media ▪ The tissue block should be 2-4mm thick (to minimize the risk of the knife hitting the metal tissue block holder)   ▪ Only cut individual sections and do not form ribbons as in paraffin blocks   ➢Fresh frozen tissues requires: • That the tissue must be maintained in the frozen solid state during cutting of section (thereby supporting and protecting the tissue from damage and distortion by the knife during the process of cutting) • Tissue must be sufficiently cold and hard (to prevent compression and displacement of cell and tissue structures as the knife passes thru it, otherwise the thin section would completely melt and form a sticky distorted mass at the edge of the knife. The success of fresh tissue sectioning

depends therefore on the temperature of both the tissue and the knife 

Freezing

➢Methods:           • Liquid nitrogen    ▪ Used in histochemistry and during intra-operative procedures.    ▪ Disadvantages:        o Soft tissue is liable to crack due to the rapid expansion of the ice within the tissue, producing ice crystals or freeze artifacts        o Overcools urgent biopsy blocks, causing damage to both block and blade if sectioning is done at -70°C or below        o Causes vapor phase to form around the tissue ,acting as an insulator that causes uneven cooling of tissue, particularly of muscle biopsies, and making diagnosticinterpretation difficult (can be overcome by freezing the tissue in isopentane ,O.C.T.(Optimal Cutting Temperature compound), or Freon 2.2 that has a high thermalconductivity  • Isopentane cooled by liquid nitrogen   ▪The most rapid and liquid at room temperature • Carbon dioxide gas • Aerosol sprays   ▪ For freezing small pieces of tissue, except muscle tissue

Problems encountered during sectioning PROBLEM

CAUSE

REMEDY

Ice crystal artifacts

slow freezing of tissue

Freeze fast (flash / snap)

Knife artifact

A nicked cutting blade

change your blade every few cases or for each case

Overfreezing

Polish block with a couple extra turns of the blade to create friction and warm up block by pressing on it with your finger (5 - 10 seconds)

Dirty "stain line"

(a) Maintain a clean stain line by frequent solution changes (b) Follow recommended staining times (c) Don't rush

Section have holes

Floaters (extraneous foreign tissue) adhere to slides 

Special processing technique ➢ Methods used if chemical fixation of tissue blocks is to be avoided:  • Freeze-drying  ▪ Preserves tissues by rapid freezing (qu qu quen en enchi chi ching ng ng) of fresh tissue at -160°C and subsequently removing ice water molecules (desi desi desicca cca ccati ti tion on on) by a physical process of transferringthe still frozen tissue block in a vacuum at a higher temperature, e.g., -40°C (su su subli bli blima ma mati ti tion on on) without the use of any chemical fixative  ▪ The frozen tissue is subjected to dehydration in an expensive vacuum drying apparatus ▪ Use is restricted to specialized or research laboratories  ▪ Recommended for:              Immunocytochemistry             Fluorescent antibody studies of polypeptide and polypeptide hormones...