Fixation

• FIXATION


  First and most critical step in histotechnology
• primary goal of fixation

  preserve the morphologic and chemical integrity of the cell in as life-like a manner as possible
• second goal of fixation

  to harden and protect the tissue from the trauma of further handling, so that it is easier to cut and process for microscopy.
• practical purposes of fixation

  prevent or arrest the degenerative processes which commence as soon as a tissue is deprived of its blood supply.
• Physical methods of fixation

  heating, microwaving and cryo-preservation
• Heat fixation


  rarely used on tissue specimens

  its application being confined to smears of microorganisms
• microwave fixation


  can be regarded as a form of heat fixationis now widely practiced in routine laboratories
• Cryo-preservation


  usually in the form of freeze drying, has some applications in histochemistry but is not usually applied to diagnostic tissue specimens.
• Chemical fixation


  usually achieved by immersing the specimen in the fixative solution
• Additive fixation


  chemical constituent of the fixative is taken in and becomes part of the tissue by forming cross-links
• Examples of Additive Fixative

  formalin

  mercury

  osmium tetroxide
• Non-additive fixation


  fixing agent is not incorporated into the tissue, but alters the tissue composition and stabilizes the tissue by removing the bound water attached to H-bonds of certain groups within the protein molecule
• Example of Non-addditive Fixative

  Alcohol Fixatives
• Volume

  10-20 times the volume of tissue to be fixed.
• Hydrogen Ion Concentration


  neutral pH, in the range of 6-8

  Acidity favors formation of formalin-heme pigment that appears as black, polarizable deposits in tissue
• Temperature

  40°C for regular tissue processing

  electron microscopy and some histochemistry, the ideal temperature is 0-4°C

  Formalin heated to 60°C is sometimes used for the rapid fixation of very urgent biopsy specimens
• bone marrow continues to undergo mitosis (growth) up to 30 minutes after death when refrigerated
• Nucleic acids do not react with fixatives to any extent at room temperatures, and chemical reactions including those involved in fixation are more rapid at higher temperatures.
• Thickness of section

  1 to 2 mm2 for electron microscopy

  2 cm2 wide for light microscopy
• Brain is usually suspended whole in 10% buffered formalin for 2-3 weeks
• Formalin and alcohol penetrate the best, and glutaraldehyde the worst. Mercurials and others are somewhere in between.
• Osmolality

  fixed in a hypertonic solution, the cells may shrink

  fixed in a hypotonic solution, the cells may swell and burst

  Isotonic as well as hypotonic fixatives cause cell swelling and poor fixation (340 mOsm)

  recommend using a normal phosphate buffered saline (PBS) based fixative

  best results are usually obtained using slightly hypertonic solutions (400-450 mOsm)
• Concentration

  adjusted down to the lowest level possible

  Too high a concentration may adversely affect the tissue and produce artefact similar to that caused by excessive heat
• Duration of fixation

  Fibrous organs such as uterus or intestinal tract take longer to fix than small or loosely textured tissues such as biopsies or scrapings
• Prolonged fixation may cause shrinkage and hardening of tissue, and may severely inhibit enzyme activity and immunological reactions, although washing of the tissue in running water considerably restores the activity of some enzymes
• electron microscopy, it is recommended that diced tissues be fixed for 3 hours and then placed in a holding buffer.
• Time Interval


  fixed immediately after removal or death to prevent autolysis or putrefaction
• Simple Fixatives


  made up of only one component substance
• Compound Fixatives


  made up of two or more fixatives which have been added together to obtain the optimal combined effect of their individual actions upon the cells and tissue constituents.
• Microanatomical Fixatives


  permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues in question
• Microanatomical Fixatives


  10% formal saline

  10% neutral buffered formalin

  Heidenhain 's Susa

  Formal sublimate (formal corrosive)

  Zenker 's solution

  Zenker-formal (Kelly 's solution)

  Bouin's solution

  Brasil's solution
• Cytological Fixatives


  preserve specific parts and particular microscopic elements of the cell itself.
• Nuclear Fixatives


  preserve the nuclear structures (e.g., chromosomes) in particular. They usually contain glacial acetic acid as their primary component due to its affinity for nuclear chromatin.

  They have a pH of 4.6 or less.
• Nuclear Fixative


  Flemming's fluid

  Carnoy's fluid

  Bouin's fluid

  Newcomer's fluid

  Heidenhain's Susa
• Cytoplasmic Fixatives


  preserve cytoplasmic structures in particular.

  They must never contain glacial acetic acid which destroys mitochondria and Golgi bodies of the cytoplasm.

  They have a pH of more than 4.6
• Cytoplasmic Fixatives


  Flemming's fluid without acetic acid

  Kelly's fluid

  Formalin with "post-chroming"

  Regaud 's fluid (Muller 's fluid)

  Orth 's fluid
• For RNA

  the precipitant fixatives - ethanol and acetone - give the best quantitative results using frozen tissues as the standard
• Histochemical Fixatives


  those that preserve the chemical constituents of cells and tissues.
• Histochemical Fixatives


  Formal Saline 10%

  Absolute Ethyl

  Newcomer's Fluid

  Alcohol Acetone
• Secondary Fixation


  process of placing an already fixed tissue in a second fixative in order:

  To facilitate and improve the demonstration of particular substances.

  To make special staining techniques possible (with secondary fixative acting as a mordant).

  To ensure further and complete hardening and preservation of tissues.