Cellular pathology

Cellular pathology

1. Basic histology

   • Oragns composed of cells within an intercellular matrix of solid fibres and a gelatinous or liquid ground substance
     • The cells synthesise and maintain this intercellular matrix
       • This matrix provides
         • Physical support
         • Protection
         • A pathway for diffusion to and from each cell
   • Histology
     • Study of the tissues and their strcutures
     • Cellular pathology
       • Study of diseass in tissues and whole body organs
     • Tissues first have to be processed - fixed, sectioned and then stained
       • Artefacts are alterations to the tissue caused by processing

2. Processing

   • Fixation
     • Dehydration
       • Clearing
         • Removal of the dehydrating agent
           • Using toluene, xylene, chloroform, 1,1,1-trichloroehtane (all wax miscible)
         • Impregnation with molten wax (embedding)
           • Tissue is usually too soft to be cut into 3-6um sections
             • A hard embedding medium is used to support the tissue
               • This step prepares for microtomy
           • Process
             • Paraffin wax (melted at 60 degressC)
               • After dehydration the dehydrating agent is replaced with an agent miscible with wax (e.g. toluene)
           • Blocking out (parafin wax containing the tissue specimen is shaped into a manageable block)
             • Microtomy
               • Cuts wax-embedded tissue into regular 5um sections (ideal for light microscopy)
               • Types
                 • Handheld
                   • Small and simple
                 • Rotary
                   • Most common lab microtome - produces ribbon of section
                 • Sliding
                   • Cuts larger blocks
                 • Sledge
                   • For whole organs (e.g. lung)
               • Mounting (to a slide)
                 • Staining
             • Using moulds
             • At this point it is important to think about the orientation of the tissue and what you're trying to see
           • Other embedding materials
             • Plastics
               • Acrylic
                 • Light microscopy
               • Epoxy
                 • EM
         • Factors affecting clearing
           • Speed of dehydrating agent replacement
           • Speed of evaporation of clearing agent from tissue in wax bath
           • Shrinkage and hardening
           • Cost, safety
       • Removes water and allows for replacement in parafin wax (embedding)
       • Uses solvents - e.g. ethanol 9commonest), methanol, acetone
       • Factors affecting selection
         • Cost
         • Speed of dehydration
           • Dependent on the viscosity of the solvent
         • Safety
         • Affects on tissues
     • To avoid tissue degenration
       • Mechanism of degeneration
         • Putrification
           • Caused by microbes
         • Autolysis
           • Lysosomal enzymes
         • Abnormal metabolism
           • In isolated tissues
     • Preserve tissues in a life-like condition
       • Typically uses enzyme poisons
     • Ideal properties
       • Penetrates tissues quickly
       • Doesnt damage/kill cells
       • Prevents autolysis
       • Prevents putrefaction
       • Doesnt leave artefacts
       • Doesn't distort tissues
       • Allows for staining
       • Non toxic and cheap
     • Different fixatives
       • Formaldehyde (most common) - causes crosslinks in proteins
       • Glutaraldehyde
       • Acetic acid
       • Osmium tetroxide
       • Zenker's fixative

3. Staining

   • To give a contrast between different tissue components
     • Then examine
   • Bonding occurs between the dye and the tissue
   • Dyes
     • Ionic, coloured, organic molecules
   • Mordants
     • Metallic salts that help dyes to bind
   • Haematoxylin and eosin
     • The standard tissue dye
       • Haematoxylin
         • Stains nucleic acids (plus clacium deposits in bacteria) blue
       • Eosin
         • Stains most proteins (Arg and Lys residue) pink
       • Clear areas represent; water, carbohydrates, lipids or gas
   • Metallic staining
     • E.g. using silver salts
   • Histochemical stains
     • Van Gieson
       • Stains collagen and aids differentiation of connective tissues
     • Gomori non-specific alkaline phosphatase stain
       • Highlights tissue phosphatase enzymes (e.g. in liver)