HPCT STAINING, PRINCIPLES, OTHERS

• Staining


  The process of applying dyes on the sections to see and study the architectural pattern of the tissue and the physical characteristics of the cell

  
• Aims of Staining


  • Make the cell structure visible
  • Show variation in structure; normal and abnormal
  • Indicate the chemical nature and tissue entities; nucleus or cytoplasm

  
• Differential action of dyes


  The depth of colouration is affected by: Chemical affinity, Density, Permeability + diffusion of dye
• Hematoxylin stains nuclear details, Eosin stains cytoplasmic
• Types of Tissue Staining

  • Histological Staining
  • Histochemical Staining (Histochemistry)
  • Immunohistochemical staining
• Histological Staining


  A process whereby the tissue constituents are demonstrated in sections by direct interaction with a dye or staining solution producing coloration of the active tissue component

  
• Histological Staining Types


  • Micro-anatomic stains
  • Bacterial stains
  • Specific tissue stains (e.g. muscles, connective tissue and neurologic stains)
• Histochemical Staining (Histochemistry)


  A process whereby various constituents of tissues are studied through chemical reactions that will permit microscopic localization of a specific tissue substance

  
• Histochemical Staining Examples


  • Perl's prussian blue for hemoglobin
  • Periodic Acid Schiff for carbohydrates

  
• Immunohistochemical staining


  A combination of immunologic and histochemical techniques that allow phenotypic markers to be detected and demonstrated under the microscope, using a wide range of polyclonal or monoclonal, fluorescent labeled or enzyme-labeled antibodies
• Methods of Staining


  • Direct Staining
  • Indirect Staining
  • Progressive Staining
  • Regressive Staining
  • Differential Staining

  
• Direct Staining


  The process of giving color to the sections by using aqueous or alcoholic dye solutions (e.g.,methylene blue, eosin)

  
• Indirect Staining


  A process whereby the action of the dye is intensified by adding: Another agent or Mordant, Accentuator

  
• Mordant


  Serves as a link or bridge between the tissue and the dye to make the the staining reaction possible

  
• Mordant Examples


  • Potassium alum with hematoxylin in Ehrlich's hematoxylin
  • Iron in Weigert's hematoxylin
• Accentuator


  Participation is not essential to the chemical union, does not participate in the staining reaction but merely accelerates and hastens the speed of staining reaction by increasing the staining power and selectivity of the dye
• Accentuator Examples


  • Potassium hydroxide in Loeffler's methylene blue
  • Phenol in carbolthionine and carbol fuchsin
• Progressive Staining


  Process whereby tissue elements are stained in a definite sequence and the staining solution is applied for specific periods of time or until the desired intensity of coloring of the different tissue elements is attained

  
• Regressive Staining


  Tissue is first overstained to obliterate the cellular details and the excess stain is removed or decolorized from unwanted parts of the tissue until the desired intensity of color is obtained

  
• Differentiation (Decolorization)


  A selective removal of excess stain from the tissue during regressive staining in order that a specific substance may be stained distinctly from the surrounding tissues

  
• Differential Staining


  Uses more than one chemical stain, usually done by washing the section in simple solution (e.g. water or alcohol), or by the use of acids and alkaline solutions

  
• If primary stain used is a basic dye, differentiation is carried out by an acid solution. If primary stain used is an acidic dye, differentiation is carried out by an alkaline solution

  
• Differential staining is also used to detect abnormalities in the proportion of different white blood cells in the blood (WBC Differential)
• Metachromatic Staining


  It entails the use of specific dyes which differentiate particular substances by staining them with a color that is different from that of the stain itself (metachromasia)

  
• Metachromatic Dyes


  • Methyl violet or crystal violet
  • Cresyl blue (for reticulocytes)
  • Safranin
  • Bismarck brown
  • Basic fuchsin
  • Methylene blue
  • Thionine
  • Toluidine blue
  • Azure A, B, C
• Azures or toluidine blue are more effective for metachromatic staining, except for amyloid which shows significant metachromasia with crystal or methyl violets

  
• Counterstaining


  An application of a different color or stain to provide contrast and background to the staining of the structural components to be demonstrated

  
• Cytoplasmic Stains

  • Red: Eosin Y, Eosin B, Phloxine B
  • Yellow: Picric Acid, Orange G, Rose Bengal
  • Green: Light Green SF, Lissamine Green

  
• Nuclear Stains


  • Red: Neutral Red, Safranin O, Carmine, Hematoxylin
  • Blue: Methylene Blue, Toluidine Blue, Celestine Blue

  
• Metallic Impregnation


  A process where specific tissue elements are demonstrated, not by stains, but by colorless solution of metallic salts producing an opaque, usually black deposits, on the surface of tissue or bacteria
• Most Valuable Metals

  • Gold (Gold Chloride) and Silver (Silver Nitrate)
• Vital Staining


  Selective staining of living cell constituents, demonstrating cytoplasmic structures by phagocytosis of the dye particle (cytoplasmic phagocytosis)

  
• The nucleus of a living cell is resistant to vital stains, demonstration of which leads to the death of the cell
• Intravital Staining


  Staining of living cells by injecting the dye into any part of the body (intravenous, intraperitoneal, or subcutaneous) producing specific coloration of the reticulo-endothelial system

  
• Common Intravital Dyes

  • Lithium
  • Carmine
  • India Ink
• Supravital Staining


  Staining of living cells immediately after removal from the body
• Common Supravital Dyes


  • Neutral Red
  • Janus Green
  • Trypan Blue
  • Nile Blue
  • Thionine
  • Toluidine Blue

  
• Categories of Dyes


  • Natural dyes - cochineal dyes, logwood dyes, and vegetable extracts
  • Synthetic dyes (artificial dyes) – aniline or coal tar dyes
• Hematoxylin


  Derived by extraction from the core or the heartwood of a Mexican tree "Hematoxylin Campechianum", has powerful nuclear and chromatin staining capacity, and striking polychrome properties

  
• Cochineal Dye


  An old histologic dye extracted from the female cochineal bug (Coccus Cacti), which is treated with alum to produce the dye, carmine