Histopathology

Histopathology

1. wear gloves, labelled container and corresponding request form in sealed bag.

   • Fixation - preserves tissue in as life-like state as poss, & complements & enhances subsequent staining & immunohistochem procedures
     • factors affecting optimal fixation
       • 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
         • 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
       • The ideal fixative
         • 1. penetrates quickly & evenly
         • 2. Kills cells quickly & evenly
         • 3. Prevents autolysis
         • 4. Prevents putrefaction
           • decomposition
         • 5. Does not swell, shrink or harden tissue
         • 6. Stabilizes the tissue against processing trauma
         • 7. Prepares tissue for further treatment,eg. staining
         • 8. Safe to use non-toxic / non-flammable
         • 9. Convenient to use (shelf life, storage, etc)
     • How Fixatives Work
       • all react with proteins in 1 of 2 ways:
         • Non-coagulants - form cross link with tissue protein, eg. formalin
         • Coagulants - disrupt protein structure, eg. alcohol acetone
     • Types of Fixative
       • Simple
         • Single chemical solution - no additives, e.g. methanol, ethanol, glacial acetic acid & formalin, disadvantage - may produce artefacts
       • Compound
         • Mixture of simple fixatives, eg. Carnoy's fluid (nucleic acids), 60% ethanol + 30% chloroform + 10% acetic acid

2. Flow of specimen in lab

   • Receptoon, fixation, dehydration, clearing, wax infiltration, embedding, microtomy, staining, mounting, microscopical examination
     • Tissue Processing
       • “The treatment of tissues by fluids, leading to impregnation by solidifiable embedding medium" PROFESSOR AW CURRIE (1988)
         • = Go from aqueous fixative to wax
       • Commonly used with paraffin wax embedding
         • Alternatives: waxes, resins, gelatine, celloidin & agar
       • 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.
       • Good Tissue Processing
         • 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
       • Bad Tissue Processing
         • Tissue is crammed into the processing cassette = prohibits adequate circulation of reagents
       • Stages
         • 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.
       • Factors affecting tissue processing
         • 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.
         • 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.
         • 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.
     • Microtomy
       • Cutting biological specimens into very thin segments for microscope examination. Mechanical microtome is used. Most common is Rotary microtome. (Ribbons of 4um thick)
       • Considerations - how big is the block, how hard, are serial sections needed, cost
       • Then float ribbon in water of flotation bath, and collect ribbon onto centre of a slide.
         • H&E staining next (common)

3. Class of Fixatives

   • Aldehydes - formlain & glutaraldhye
     • Formalin
       • Common fixative, 40% soluble in water (40% formaldehyde in solution of 100% formalin)
       • non-buffered solution will become acidic => formic acid (reacts with blood rich tissues, e.g. spleen = black pigment - acid formation haematin)
       • forms cross links between lysine residues, soluble proteins are fixed to structural proteins - gives strength to tissue to allow subsequent processing
         • Does NOT fix carbohydrates, but DOES fix glycogen which is then fixed to proteins, reacts with lipids but does not stabilise them
       • Advantages
         • 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.
       • Disadvantages
         • 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
   • Denaturing agents - alcohol, ethanol, methanol
     • Simple (alcohols)
       • Advantages
         • Pens tissues rapidly, may be used together with other fixatives to increase the speed of fix, preserves glycogen
       • Disadvantages
         • 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.
     • Compound (Carnoy's Fluid)
       • Ethanol + Chloroform + acetic acid
       • nucleic acids
       • Causes shrinkage of tissue, and lysis of RBCs
   • Oxidising Agents - Osmium tetroxide & potassium dichromate
     • Secondary fixative for EM analysis (after primary fixation with glutaraldehyde), Good fixative for lipids, preserves fine structures of the cell
     • Toxic - safety precautions, expensive, causes distortion & shrinkage
     • Osmium tetroxide
     • Potassium Dichromate
       • Not suitable as a general fixative - due to slow pen of tissue, causes tissue shrinkage
       • Identify adrenal medullary tumours both macro & microscopically, reacts with adrenal medullary catecholamines to produce
   • Additives
     • Tannic acid, phenol, heavy metal
     • may all be added to formalin to increase pen rate, improves preservation, enhance subsequent staining
   • Vapour Fixation
     • 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
     • Microwave ovens may be used to preserve tissue using heat, or speed up the process of a fixative solution

4. Frozen Sectioning

   • Used for rapid diagnosis of suspect tumour.
   • Cryostat in refridgerated chamber used to section tissue.
   • 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.

5. EM

   • Requires the production of ultra thin resin embedded sections using ultramicrotome
   • Hard epoxy or araldite resins are usually employed
   • Section thickness in namometres (60-90nm), knives are glass or diamond
     • interference colour when section in contact with water: Gold (90-120 nm) Silver (60-90 nm), Grey (<60nm)
   • Staining Sections - sections picked up & stained on special copper &/or rhodium grids
     • Stains: Heavy metals (lead citrate or uranyl acetate), Impregnate certain tissue stuctures, Heavy metals deflect an electron beam produced by EM.
   • Image Analysis
     • Shades of black & grey areas within the tissue section where ultrastructural elements have been impregnated by the heavy metals.
     • EM has camera system, areas of interest within section may be photo = Electron Micrograph

6. Differences between Paraffin Wax & EM

   • Paraffin Wax - Thickness um, embed in plastic cassettes, steel knives, stained with dyes, light, glass slide
   • Epoxy Resin, Thickness nm, Collect in copper &/or rhodium grids, diamond knives, metal stains, electrons, micrograph