HISTO-Decalcification

• Decalcification


  The removal of calcium ions from a bone or calcified tissue through a histological process that makes them flexible and easier to cut
• Decalcification


  • Adjusts the hard substance of bones to the softness of paraffin embedding medium
  • Enables the histotechnologist to cut soft sections of the bone using the microtome, so that they can be processed like any other soft tissue of the body
• Objects of decalcification


  • Bones
  • Teeth
  • Calcified tumors
  • Calcified heart valves
• Fine detail radiographs are often used to assist in the selection of appropriate bone specimens for processing
• If the calcified areas in tissue specimens are substantial, it may be impossible to obtain decent sections without first decalcifying the entire specimen
• Surface decalcification


  Applying decalcification to a paraffin block, allowing sections to be obtained where the presence of calcium was not anticipated when the specimen was processed
• Decalcification is a lengthy procedure, as bone pieces have to be left in the decalcifying agent for several days or even weeks, depending on the size of the tissue
• A low speed saw may be sufficient to routinely and rapidly reduce undecalcified surgical specimens of hard tissue, to a thickness of 2–3 mm, without compromising the integrity of the tissue
• Un-decalcified bone sections


  Necessary to differentiate mineralized bone from osteoid, or if morphometric measurements are required, in order to retain and demonstrate the mineral content
• Producing un-decalcified bone sections

  1. Fixation

  2. Directly sawn into thin wafers

  3. Ground using abrasive surfaces to produce thin "ground" sections
• Principle of decalcification


  • Strong mineral acids, such as 10% hydrogen chloride (HCl), or weak organic acids, such as 5-10% formic acid (HCOOH), form soluble calcium salts in an ion exchange that moves calcium into the decalcifying solution
  • 14% ethylene diamino tetracetic acid (EDTA) is an ideal chelating agent that sequesters metallic ions, including calcium, in aqueous solutions
• Buffered formalin is a satisfactory fixative for bone but where the preservation of bone marrow is important, some laboratories use alternatives such as zinc formalin mixtures, B-5, formol-acetic alcohol (Davidson's fixative), or Bouin's solution
• Decalcification should be done after fixation and before impregnation, to ensure and facilitate the normal cutting of sections and to prevent obscuring the microanatomic detail of such sections by bone dust and other cellular debris
• Inadequate decalcification may result in poor cutting of hard tissues and damage to the knife edge during sectioning
• Poorly-fixed specimens become macerated during decalcification and stain poorly afterwards
• Cartilage does not require any softening, except if some calcified areas are present
• It is a waste of time to put toenails in decalcification solution, because they are composed of insoluble keratin filaments
• Types of decalcifying agents

  • Those based on strong mineral acids
  • Those based on weaker organic acids
  • Those composed of chelating agents
• Acids


  Make up a solution of calcium ions
• Chelating agents


  Take up the calcium ions
• Nuclear and cytoplasmic detail are compromised if specimens are exposed for too long to acidic decalcifying agents, which can extract RNA and remove the purine and pyrimidine bases from DNA
• It is imperative to wash the acid out of the tissue
• Acid decalcifying agents


  • They are the most widely used agents for routine decalcification of large amounts of bony tissues because they are stable, readily available, and relatively inexpensive as compared to other decalcifying agents
• Decalcification procedure


  1. Fixation complete

  2. Tissue pieces placed in gauze bag and suspended in liberal amounts of decalcifying solution

  3. Use of thread dipped in melted paraffin wax to avoid corrosive action of acid

  4. Avoid metal cap containers
• Strong mineral acids

  Hydrochloric or nitric acid at concentrations up to 10% are the most rapid in action but if used longer than necessary will rapidly cause a loss of nuclear staining and can macerate tissues
• Rapid decalcifying agents are more likely to adversely affect any subsequent staining, especially in cell nuclei due to failure of nuclear chromatin to take up hematoxylin and other basic dyes as readily as soft tissues that have not been exposed to acid solutions or decalcifiers
• Nitric acid


  The most common and the fastest decalcifying agent used so far, utilized both as a simple solution or combined with other reagents
• Aqueous Nitric Acid Solution 10%


  • Advantages: Rapid in action, produces minimum distortion, good nuclear staining, acid easily removed, recommended for urgent biopsy

  Disadvantages: Prolonged decalcification may lead to tissue distortion, can seriously damage tissue stainability, imparts yellow color
• Formol-Nitric Acid


  • Advantages: Rapid-acting, relatively good nuclear staining

  Disadvantages: Yellow color imparted by nitrous acid formation, solution should be used inside a fume hood
• Perenyi's Fluid


  • Advantages: Recommended for routine purposes, decalcifies and softens tissues, good nuclear and cytoplasmic staining

  Disadvantages: Slow for dense bones, complete decalcification cannot be determined by chemical test
• Phloroglucin-Nitric Acid


  • Advantage: Most rapid decalcifying agent

  Disadvantages: Poor nuclear staining, extreme tissue distortion, yellow color must be neutralized
• Hydrochloric acid (HCl)


  Inferior to nitric acid as a decalcifying agent due to slower action and greater distortion, but produces good nuclear staining
• Von Ebner's Fluid


  • Advantages: Permits relatively good cytologic staining, moderately rapid, does not require washing out before dehydration

  Disadvantage: Extent of decalcification cannot be measured by a chemical test
• Weak acids


  Such as formic acid, are better suited to bone marrow, since they are not as harsh, but act more slowly on dense cortical bone
• Picric acid and acetic acid are not used alone as decalcifying agents, but are found as components of Carnoy's and Bouin's fixatives, which may act as incidental, albeit, weak decalcifiers, and can be used in urgent cases when there is insufficient time for complete decalcification
• Strong mixture


  Considered too concentrated
• Von Ebner's Fluid


  • Saturated aqueous solution of NaCl 50 ml
  • 36% concentrated hydrochloric acid 8 ml
  • Distilled water 50 ml
• Advantages of Von Ebner's Fluid

  • It permits relatively good cytologic staining
  • It is a moderately rapid decalcifying agent
  • It does not require washing out before dehydration
  • It is recommended for teeth and small pieces of bone
• Disadvantage of Von Ebner's Fluid

  The extent of decalcification cannot be measured by a chemical test
• Weak acids


  Such as formic acid, are popular and widely used for decalcification