cyto3

• Pigments


  Substances that give colour to tissues
• Classification of pigments


  • Artefact pigments
  • Exogenous pigments
  • Endogenous pigments
• Artefact pigments


  • Usually as a result of fixation
  • Normally lie on top of tissues and not within cells
  • Brown-black deposits
• Artefact pigments


  • Formalin
  • Mercury
  • Chrome
  • Picrate's
• Formalin pigment


  • Result of fixation of tissue in acid formalin
  • Also called acid haematin
  • Brown-black deposits
  • Resistant to strong acids
  • Usually associated with blood-containing tissues
  • Can be removed from sections using alcoholic picric acid
• Mercury pigment


  • Occurs with mercury-containing fixatives such as Zenker's and Heidenhain's Susa
  • Dark brown-black granules
  • Can be removed from sections using iodine followed by sodium thiosulphate (hyposulphite) solution
• Chrome pigment


  • Yellow deposit
  • Consequence of fixation in solutions containing potassium dichromate eg Zenker's fixative
  • Can be removed from sections by thoroughly washing in water prior to dehydration (or 1% acid alcohol)
• Picrate pigment


  • Consequence of fixation in solutions containing picric acid eg Bouin's fixative
  • Can be removed with saturated aqueous lithium carbonate
• Exogenous pigments


  Pigments that are formed externally and taken into the body
• Exogenous pigments


  • Carbon
  • Tattoo
• Asbestos


  Become coated with iron in the lungs (bodies) and stain positive with Perl's
• Minerals


  • Metallic and non-metallic ions necessary for growth and other bodily functions
  • Calcium, ferrous and ferric iron, copper, phosphate and carbonate are the most common
  • Some can be deposited pathologically in tissue eg: gold, silver, copper and lead
• Microincineration


  1. Cut paraffin sections as usual and mount on high temperature glass slides

  2. Slides are incinerated up to 650oC and then allowed to cool

  3. Slides are removed from oven and coverslipped with glycerol mountant

  4. Slides are examined under the microscope
• Calcium


  • Abnormal deposits can be associated with atherosclerosis, sarcoid, TB and some tumours
  • Also found in joints in chondrocalcinosis
  • Demonstrate with von Kossa silver method
• Von Kossa method


  • Indirect way of detecting calcium where silver nitrate replaces the carbonate anions of calcium salts (silver substitution method)
  • Sunlight reduces the silver salt to visible metallic silver, and unreduced silver is removed by sodium thiosulphate
• Von Kossa method


  • Calcium salts black, Nuclei red, Cytoplasm pink
• Copper


  • Accumulates in liver and other organs in Wilson's disease
  • Also found in primary biliary cirrhosis and other liver disorders
  • Stain with rhodanine (or rubeanic acid)
• Rhodanine method


  • Rhodanine demonstrates the protein to which the copper binds rather than the copper itself
  • Copper deposits - brick red, Nuclei - pale blue, Bile - green
• Haematogenous pigments


  • Endogenous pigments derived from blood
  • Main ones are haemoglobin, iron, haemosiderin, haematoidin and bile pigments
• Haemosiderin


  • Stored as ferric iron in bone marrow and spleen
  • Can be demonstrated using Perl's prussian blue stain which detects ferric iron
  • Excessive absorption of dietary iron causes haemochromatosis
  • Ferrous iron is demonstrated by Turnbull's blue
• Perl's stain


  • Ferric iron is released from haemosiderin with hydrochloric acid, forming ferric chloride
  • The iron reacts with potassium ferrocyanide to form ferric ferrocyanide (Prussian blue) (bright blue pigment)
• Turnbull's blue stain


  • Treatment with acid solution of potassium ferricyanide
  • Any ferrous iron present reacts to form an insoluble bright blue pigment called Turnbull's blue (ferrous ferricyanide)
• Bile and bilirubin


  • Biliverdin (green) is transported to the liver
  • Undergoes reduction to bilirubin (yellow-brown)
  • Can be demonstrated by Hall's method which uses Fouchet's reagent
• Hall's method


  • A specific and easily identifiable green colour develops when bilirubin is oxidized to biliverdin in an acid medium
  • This oxidation reaction is rapidly accomplished by ferric chloride in trichloroacetic acid medium (Fouchet's reagent)
  • Biliverdin is then stained emerald green by acid fuchsin in van Gieson solution
• Non-haematogenous pigments


  • Endogenous pigments not derived from blood
  • There are two types: Lipidic (lipofuschin and ceroid) and Non-lipidic (melanin)
• Lipofuschin


  • Breakdown product of lipids and lipoproteins
  • Usually found in the heart and liver around the nucleus
  • Stain with Schmorl's stain, oil red O, aldehyde fuchsin, Sudan black B and PAS
• Melanin


  • Found in skin, hair, retina and parts of the CNS
  • Can be bleached in the tissue section by treatment with hydrogen peroxide or potassium permanganate and oxalic acid
  • Pathologically, melanin is found in the cells of malignant melanomas and various benign naevi
  • Melanin reduces silver nitrate to metallic silver in the Masson Fontana method (argentaffin)
• Lillie's sulfuric nile blue method

  Melanin - dark green, Lipofuscin - dark blue or blue-green, Nuclei and background - blue