wk 11- intro to cell path

• Cellular Pathology


  The study of disease within tissues & cells
• Histology


  • The study of tissues
• Cytology


  • The study of cells
• Histopathology


  • The study of disease within tissues
• Cytopathology


  • The study of disease within cells
• Tissues/cells prepared by specific laboratory techniques so that they can be viewed down the microscope
• Enables suspected diagnoses from other depts/tests to be confirmed
• Consultant Histo/Cytopathologists


  • Vital to Cellular Pathology
  • Provide diagnoses, treatment options or CoD (PMs)
• Main types of sample which come to the Cellular Pathology laboratory

  • Small tissue samples (such as biopsies & needlecores)
  • Medium (such as skin samples, lumpectomies)
  • Large tissue samples (resections/entire organs)
  • Cytological samples (such as sputum, pleural effusions, FNAs)
• Small tissue samples


  • Endoscopic - colon, oesophagus, bronchial, any accessible luminal surface
  • Needle biopsy – liver, lung, breast, prostate – any solid organ accessible to a needle
  • Scrapes / curetting – epithelium mechanically removed, e.g. skin or endometrial curetting
• Medium tissue samples


  • Incisional – a small sample of the lesion, with adjacent unaffected tissue is taken (diagnostic)
  • Excisional – the entire lesion or organ is removed, with a clear margin of unaffected tissue (curative)
• Large tissue samples


  • Large resections
  • Whole organs
  • Benign
  • Malignant
  • Removes diseased tissue and affected organ
• Cytological samples


  • Fluids can be centrifuged to concentrate cellular material
  • Narrow gauge needle used to draw out a sample of cells, which are then spread on a slide
• Histology techniques


  1. Fixation

  2. Dissection/tissue transfer

  3. Processing

  4. Embedding

  5. Sectioning

  6. Staining
• Fixation


  Stops cellular breakdown, autolysis, putrefaction, aims to preserve the tissue in as life like a state as possible
• Fixatives


  • Simple (Alcohol e.g. Methanol, Ethanol, Formaldehyde)
  • Compound (Carnoy's, Zenker's, Bouin's)
  • Formaldehyde (formalin)
• Freezing


  • Only tissues whereby subsequent tests incompatible with chemical fixatives e.g. Muscle biopsies (enzyme histochemistry), Renal/skin (Immunofluorescence IMF), Samples for cytogenetics
• Fixatives – not only preserve, but make easier to be stained by dyes
• Dissection/Tissue transfer


  1. Tissue Transfer

  2. Larger specimens need to be dissected
• Processing


  1. Replace water with wax

  2. Formalin to IMS to Xylene to Wax

  3. Wax provides a suitable supporting medium for sectioning
• Embedding


  1. Cassettes of tissues removed from processor in molten wax

  2. Placed in heated section of embedder

  3. Tissue orientated in a mould of molten wax

  4. Labelled cassette placed on top

  5. Placed on cold place to solidify
• Sectioning


  1. Thin (2-4µm) sections are cut from the wax blocks on a microtome

  2. Thin enough to permit light to pass through

  3. Sections are ribboned
• Tissue sectioning


  1. Ribbon of sections floated out on a warm water bath

  2. Minimises creases

  3. Picked up onto glass slides
• Frozen sections


  • Immediate, intraoperative diagnosis
  • May be to assess margins or confirm tissue type
  • Pathologist or BMS describes the sample and freezes it
  • Tissue cut on a cryostat– temperature controlled microtome
• Staining: The H&E


  • Tissue processed into wax is fairly colourless
  • Staining provides contrast for light microscopy
  • Standard routine stain in Histopathology is the H&E (Haematoxylin & Eosin)
  • Most specimens can be diagnosed using an H&E only!
• Haematoxylin


  A basic dye which stains basophilic (acidic) nuclei of cells dark blue/purple
• Eosin


  An acidic dye which stains acidophilic (basic) structures such as cytoplasm, collagen, RBC and muscle red/pink/orange
• Types of "special" stain

  • Tinctorial - Use compounds to selectively stain cells and cellular components
  • Histochemical - Utilise specific chemical reactions between chemicals and tissue components
• Tinctorial stain example


  • Martius Scarlett Blue
• Histochemical stain example


  • Perl's Prussian Blue for Iron
• Hundreds of different special stains are commonly used in clinical histopathology to detect bacteria/fungo, CHOs, connective tissues and pigments/minerals
• Specialised Cell Path staining techniques/ methods

  • Immunohistochemistry
  • Immunofluorescence
  • FISH
  • PCR
• Immunohistochemistry (IHC/ICC)


  Used to detect antigens of interest on a cell or nuclear membrane or in the cytoplasm which cannot be seen by H&E alone
• The 'indirect' IHC technique

  1. Dewax sections in xylene and rehydrate in alcohol

  2. Block endogenous peroxidase

  3. Perform HMAR/enzyme digestion if required by antibody of interest

  4. Primary antibody applied ->wash off excess

  5. Labelled secondary antibody applied -> wash off excess

  6. Chromogen applied -> wash off excess

  7. Counterstain with Hx -> wash off excess

  8. DCM (dehydrate, clear and mount
• There are times when H&E alone is insufficient for accurate/definitive diagnosis
• Supplemented by ancillary staining techniques such as special stains or IC
• Antibodies can be requested individually or in panels

  Panels can be particularly useful in discriminating between different cancer types
• Examples of use of individual antibodies

  • Infective agents not easily demonstrated by special stains e.g. Hep B, HPV, EBV
  • Specific cellular structures and components (collagens, cytokeratins, actin & myosin) and their relation to cancer cells e.g. basement membrane breaches using Collagen IV / vascular invasion using D240
  • Substances produced by cells e.g. mucins, enzymes, hormones (assessment of increase/decrease) e.g. Calcitonin production by thyroid
  • Antigens in/on cells found to be linked to cancer (e.g. CA125 – ovarian cancer antigen)
  • Cellular processes such as proliferation rates (e.g. Ki67)
• Examples of IHC for prognosis

  • Breast cancer - Express ER and PR (nuclear antigens), If positive, patient will respond to Tamoxifen
  • Breast cancer - May express HER2 (membranous antigen), If positive, patient will respond to Herceptin
• Immunofluorescence (IMF)


  • Commonly performed on tissue sections (Direct IMF) or using patients' serum (Indirect IMF)
  • Usually on frozen sections
  • Procedure similar to IC but label is fluorescent, not chromogenic
  • Access to a fluorescence microscope needed
  • Stained sections can only be kept for 1-2 months (refrigerated in dark)
  • Images usually taken and stored digitally for a permanent record