Fixatives in Histopathology

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  • 1. FIXATIVESFIXATIVES Dr.Ishwarya.S II yr post graduate
  • 2. Definition of FixationDefinition of Fixation  A chemical process by which biological tissues are preserved from decay, either through autolysis or putrefaction It terminates any ongoing biochemical reactions, and may also increases the mechanical strength or stability of the treated tissues.
  • 3. Aims and Effects of fixationAims and Effects of fixation  Inhibition of Autolysis and putrefaction  Preservation  Hardening  Solidification of colloid material  Effects on staining
  • 4. Features of fixativesFeatures of fixatives  Disable intrinsic biomolecules – particularly proteolytic enzymes  Protect a sample from extrinsic damage.  Increase their mechanical strength & rigidity.
  • 5. Heat fixation Perfusion Immersion Methods of FixationMethods of Fixation
  • 6. Types of fixationTypes of fixation Physical methods of fixation Chemical methods of fixation
  • 7. Physical MethodsPhysical Methods Heat Fixation Microwave fixation - Glyoxal based fixatives Freeze drying and freeze substitution
  • 8. Chemical FixationChemical Fixation  Coagulant Fixatives  Non - coagulant cross linking fixatives
  • 9. Coagulant FixativesCoagulant Fixatives  Alcohols (Ethanol, Methanol)  Acetone  Zinc Salts  Mercuric Chloride  Chromium Trioxide  Picric acid
  • 10. Non Coagulant FixativesNon Coagulant Fixatives  Formaldehyde  Gluteraldehyde  Osmium Tetroxide  Potassium Dichromate  Acetic acid
  • 11. FormaldehydeFormaldehyde  10% Buffered Formalin - 100 ml of 40% Formaldehyde - 900 ml of distilled water - 4g Sodium phospatase (Monobasic) - 6.5g of sodium phosphate (dibasic)  Methylene Glycol  Cross links between protein end groups  Amino acid Lysine  Pigment Formation
  • 12. Gluteraldehyde Osmium Tetroxide Picric Acid Mercuric Chloride Metallic ions as a fixative supplement Compound fixatives
  • 13. Removal of fixation pigmentsRemoval of fixation pigments Formalin Pigments Immerse the sections in saturated absolute alcohol with picric acid for 10 mins to 3 hrs. Then wash with water. Also, a solution of 70% alcohol containing 3 mL of ammonium hydroxide for 30 minutes to 3 hours. Wash sections well in 1% acetic acid.
  • 14. Mercury Pigments  Immerse the sections in Gram or Lugol’s Iodine for 10 minutes.  Then place the section in a 5% solution of Sodium thiosulfate for 3 minutes.  Wash slides well in running water for 10 minutes.
  • 15. Melanin Pigments  Place the section in a solution of Potassium permanganate for 20 mins  Followed by a solution of Oxalic acid to remove the excess of potassium permanganate
  • 16. Factors affecting fixationFactors affecting fixation  Buffers and PH  Size of the specimens  Duration of Fixation  Temperature of Fixation  Concentration of Fixative  Osmolality of Fixatives & Ionic composition  Additives
  • 17. Rate of PenetrationRate of Penetration Fixative 4 Hrs 8 hrs 12 hrs 10% Acetic acid 3.8 mm 5 mm 5 mm 10% Formalin 2.7 mm 4.7 mm 5 mm 95% Ethanol 1.7 mm 3.5mm 5mm 7.5% Mercuric chloride 2 mm 3 mm 3.5 mm Sat. aqueous Picric acid 1 mm 1.5 mm 1.75 mm 2.5% Potassium bichromate 1 mm 1.5 mm 1.75 mm 0.7% Chromic acid 0.6 mm 1 mm 1.2 mm 4% Osmium Tetroxide 0.3 mm 0.5 mm 0.7 mm
  • 18. Choice of FixativeChoice of Fixative
  • 19. Fixation for individual tissuesFixation for individual tissues  Eyes - NBF, 48 hours  Brain  Breast - 10% NBF for minimum of 6-8 hrs  Lungs
  • 20.  Lymphoid tissue  Testis  Muscle Biopsies  Renal biopsies
  • 21. Characteristics of a good fixativeCharacteristics of a good fixative 1. It must kill the cell quickly without shrinkage or swelling 2. It must penetrate the tissue rapidly 3. It must inhibit bacterial decay and autolysis 4. Harden the tissue and render it insensitive to subsequent treatment as staining 5. It should allow tissue to be stored for long time 6. It should be simple to prepare and economical in use
  • 22. Thank youThank you