Histopathology

Histopathology

wear gloves, labelled container and corresponding request form in sealed bag.
1. Fixation - preserves tissue in as life-like state as poss, & complements & enhances subsequent staining & immunohistochem procedures
  1. factors affecting optimal fixation
    1. best pH 6.8, Density of tissue, Vol of fixative (10:1 ratio), speed of penetration - less dense = faster pen, type of fix affects pen speed, temp, time interval
      1. Over exposure = if tissues remain in fix for too long Causing
        Formalin - hardens tissues Mercuric chloride - shrinks tissues Oxidising agents - loss of peptide antigenicity
        Progressive induction of artefacts within tissue results from prolonged exposure
        preservatives may be used after the tissue has been washed
    2. The ideal fixative
      1. 1. penetrates quickly & evenly
      2. 2. Kills cells quickly & evenly
      3. 3. Prevents autolysis
      4. 4. Prevents putrefaction
        decomposition
      5. 5. Does not swell, shrink or harden tissue
      6. 6. Stabilizes the tissue against processing trauma
      7. 7. Prepares tissue for further treatment,eg. staining
      8. 8. Safe to use non-toxic / non-flammable
      9. 9. Convenient to use (shelf life, storage, etc)
  2. How Fixatives Work
    1. all react with proteins in 1 of 2 ways:
      1. Non-coagulants - form cross link with tissue protein, eg. formalin
      2. Coagulants - disrupt protein structure, eg. alcohol acetone
  3. Types of Fixative
    1. Simple
      1. Single chemical solution - no additives, e.g. methanol, ethanol, glacial acetic acid & formalin, disadvantage - may produce artefacts
    2. Compound
      1. Mixture of simple fixatives, eg. Carnoy's fluid (nucleic acids), 60% ethanol + 30% chloroform + 10% acetic acid
Flow of specimen in lab
1. Receptoon, fixation, dehydration, clearing, wax infiltration, embedding, microtomy, staining, mounting, microscopical examination
  1. Tissue Processing
    1. “The treatment of tissues by fluids, leading to impregnation by solidifiable embedding medium" PROFESSOR AW CURRIE (1988)
      1. = Go from aqueous fixative to wax
    2. Commonly used with paraffin wax embedding
      1. Alternatives: waxes, resins, gelatine, celloidin & agar
    3. Aiming to substitute the aqueous fixative with non-water miscible paraffin wax, & provide the tissue with sufficient support to allow sectioning while causing minimal damage to the tissue & knife edge.
    4. Good Tissue Processing
      1. Tiny biopsies placed in biopsy bags or cassettes, which are placed in processing cassette - tissues must be no more than 2-3mm think for rapid processing, or 3-5mm for think or overnight processing
    5. Bad Tissue Processing
      1. Tissue is crammed into the processing cassette = prohibits adequate circulation of reagents
    6. Stages
      1. 1. Dehydration, 2. Clearing, 3. Infiltration, 4. Embedding
        1. Dehydrating agents remove free & bound water molecules. Most common agent is 99.85% ethanol (purchased as 99%), others (acetone & methanol). Tissues are processed through a rising gradient of ethanol to 99% IMS (absolute ethanol)
        2. Clearing agent is miscible with paraffin wax- 2-3 changes required. Clearing agent removes dehydrating agent so that paraffin wax can infiltrate the tissue. E.g. xylene, toluene, chloroform, petroleum.
        3. Paraffin wax (or resin/agar) infiltrates and embeds tissue. Mixture of polycrystalline straight-chain hydrocarbon - MP range = 19-68C, higher MP the harder the wax. Routinely: 56-58C.
        4. Paraffin wax must be clean, filtered & held at 2-4C above MP. Once processing is complete the tissue basked is transferred to heated reservoir or free-standing bath of paraffin wax. Tissues are removed, singly, using heated forceps and placed face down in a prefilled mould of molten wax.
    7. Factors affecting tissue processing
      1. Temp - dehydration & clearing (37-46C), too low = reagents more viscous = slow disfusion, too hight = tissue shrinkage & hardening. Infiltration stage 2-3C above melting point of media will minimize adverse effect. As temp increases pen rate of fluids increases, most processing reagents are flammable.
      2. Vacuum & Pressure: both options are now available on modern enclose tissue processing machines. Vacuum enhances dehydration, clearing & infiltration. Pressure has little effect on dehydration & clearing - may affect diffusion of reagents - increased effect during infiltration.
      3. Agitation: Pack tissue cassettes loosely. Maximum surface area of tissue should be available for reagent exchange & circulation during processing. Suspend & agitate to prevent stagnation, modern machines allow rotation, up & down, side to side, or tidal flow motion.
  2. Microtomy
    1. Cutting biological specimens into very thin segments for microscope examination. Mechanical microtome is used. Most common is Rotary microtome. (Ribbons of 4um thick)
    2. Considerations - how big is the block, how hard, are serial sections needed, cost
    3. Then float ribbon in water of flotation bath, and collect ribbon onto centre of a slide.
      1. H&E staining next (common)
Class of Fixatives
1. Aldehydes - formlain & glutaraldhye
  1. Formalin
    1. Common fixative, 40% soluble in water (40% formaldehyde in solution of 100% formalin)
    2. non-buffered solution will become acidic => formic acid (reacts with blood rich tissues, e.g. spleen = black pigment - acid formation haematin)
    3. forms cross links between lysine residues, soluble proteins are fixed to structural proteins - gives strength to tissue to allow subsequent processing
      1. Does NOT fix carbohydrates, but DOES fix glycogen which is then fixed to proteins, reacts with lipids but does not stabilise them
    4. Advantages
      1. Soluble in water, rapidly pens tissue, preserves wide range of tissue, tissue may be stored for long periods without damage to nucleus, cytoplasm, morphology is maintained, tolerant & allows histochem to be done.
    5. Disadvantages
      1. Not fixative of choice for immunohistochemistry, process has to be reversed for viewing DNA, antigenicity is lost due to prolonged storage, health & safety
        Health & Safety - has pungent odour, strong eye, skin & mucous membrane, mutagen & carinogen, max exposure = 1 part/million, must wear gloves
2. Denaturing agents - alcohol, ethanol, methanol
  1. Simple (alcohols)
    1. Advantages
      1. Pens tissues rapidly, may be used together with other fixatives to increase the speed of fix, preserves glycogen
    2. Disadvantages
      1. Absolute ethanol causes distortion of nuclear detail, shrinkage of cyto, & tends to lyse RBCs, Alcohols cause too much brittleness & hardness, Methanol poisonous, Seldom used alone except in immunofluorescence or smear fix in cyto, health & safety issue.
  2. Compound (Carnoy's Fluid)
    1. Ethanol + Chloroform + acetic acid
    2. nucleic acids
    3. Causes shrinkage of tissue, and lysis of RBCs
3. Oxidising Agents - Osmium tetroxide & potassium dichromate
  1. Secondary fixative for EM analysis (after primary fixation with glutaraldehyde), Good fixative for lipids, preserves fine structures of the cell
  2. Toxic - safety precautions, expensive, causes distortion & shrinkage
  3. Osmium tetroxide
  4. Potassium Dichromate
    1. Not suitable as a general fixative - due to slow pen of tissue, causes tissue shrinkage
    2. Identify adrenal medullary tumours both macro & microscopically, reacts with adrenal medullary catecholamines to produce
4. Additives
  1. Tannic acid, phenol, heavy metal
  2. may all be added to formalin to increase pen rate, improves preservation, enhance subsequent staining
5. Vapour Fixation
  1. Volatile fixatives allow retention of soluble substances in situ, via conversion into insoluble porducts before coming into contact with solvents, often used in conjunction with freeze drying, formalin, osmium tetroxide & alcohol may be utilized this way
  2. Microwave ovens may be used to preserve tissue using heat, or speed up the process of a fixative solution
Frozen Sectioning
1. Used for rapid diagnosis of suspect tumour.
2. Cryostat in refridgerated chamber used to section tissue.
3. Sample usually transferred to lab on ice. Fresh tissue should be dissected and frozen in safety cabinet. Orientate correctly on special gum. Re-immerse in liquid N2 or spray to freeze. Place in cryostat chuck and allow to come to cryostat temp, 12-20 mins . Cut sections approx 8um. Lift sections by lowering slide slowly onto surface of section. When finished, place trimmings and tissue into 10% formalin to fix, fresh tissue = hazardous. Modern cryostats contain decontammation cycle.
EM
1. Requires the production of ultra thin resin embedded sections using ultramicrotome
2. Hard epoxy or araldite resins are usually employed
3. Section thickness in namometres (60-90nm), knives are glass or diamond
  1. interference colour when section in contact with water: Gold (90-120 nm) Silver (60-90 nm), Grey (<60nm)
4. Staining Sections - sections picked up & stained on special copper &/or rhodium grids
  1. Stains: Heavy metals (lead citrate or uranyl acetate), Impregnate certain tissue stuctures, Heavy metals deflect an electron beam produced by EM.
5. Image Analysis
  1. Shades of black & grey areas within the tissue section where ultrastructural elements have been impregnated by the heavy metals.
  2. EM has camera system, areas of interest within section may be photo = Electron Micrograph
Differences between Paraffin Wax & EM
1. Paraffin Wax - Thickness um, embed in plastic cassettes, steel knives, stained with dyes, light, glass slide
2. Epoxy Resin, Thickness nm, Collect in copper &/or rhodium grids, diamond knives, metal stains, electrons, micrograph

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	Created by sam.wats over 10 years ago