Fixation

After biopsy tissue fixation is considered as the most important process. here in this presentation various points have been elaborated.

1. Tissue fixation Presented by Dr. Shrikant Sonune Guided by Dr Ashok Patil, Dr Shilpa Kandalgaonkar, Dr Mayur Chaudhary , Dr Suyog Tupsakhare, Dr Mahesh Gabhane.

2. Content Introduction Function of fixative Methods of fixation Reaction of the cell(its component) with fixatives Simple aqueous fixatives or fixative ingredients Factors affecting fixation Effect of fixation References

3. Introduction

4. Introduction Fixation Tissue processing Sectioning Staining Staining Sectioning Tissue processing Fixation

5. Fixation : introduction Fixation is the complex series of chemical events and differs for the different groups of chemical substances found in tissues. It is most essential part of histology. Here where everything starts. Why?

6. Introduction Once the tissue is removed from the body it will go through a process of self-destruction. This process is known as autolysis. If tissue is left without any preservation, then a bacterial attack will occur, the process is known as putrefaction.

7. Definition : It is a process by which the constituents of the cells and therefore of the tissues are fixed in a physical and partly in a chemical state , so that they will withstand subsequent treatment with various reagents with minimum of loss, significant distortion or decomposition . The preservation and hardening of a tissue sample to retain as nearly as possible the same relations they had in the living body

8. Aims & objectives of fixation To prevent autolysis and putrefaction. Rapid and even penetration. To preserve cells and tissues in a life like manner as possible. Elements that are to be demonstrated must remain in maximum concentration and precise localization. Stabilize labile elements. Must be rigid to allow sectioning. Must allow staining. Optical contrast must be induced for morphological examination. Allow long storage of tissues

9. Methods of fixation 1. heat   2. Chemicals - Additive - Non additive   - Coagulant - Non-coagulant (Baker’s classification) - Coagulant • Alcohol • Zinc salts • Mercuric chloride • Chromium trioxide • Picric acid   - Non-coagulant • Formaldehyde • Glutaraldehyde • Osmium tetroxide • Potassium dichromate • Acetic acid

10. Classification of chemical fixatives 1. Aldehydes Formaldehyde, glutaraldehyde 2. Oxidizing agents Osmium tetroxide, potassium permanganate, 3. Proteins denaturing agents or coagulant Acetic acid, methyl alcohol, ethyl alcohol. 4. Other cross linking agents Carbodiimides 5. Miscellaneous Mercuric sodium, picric acid , dye stuff. (by bancroff )

11. Acc. to no. of fixatives used: - Simple fixatives - Compound fixatives i ) Micro anatomical fixatives ii) Cytological iii)Histochemical

12. Reaction of fixatives with Protein Most important reactions which stabilizes proteins by forming cross links between soluble protein & structural protein. Ultimately providing some mechanical strength.

13. Aldehydes Cross links are formed between protein molecules and Aldehyde group of fixative. Aldehydes react with the basic amino acid residues of proteins & there is an accompanying change in isoeletric point of proteins. This may form the basis for the of the different staining of tissues after different fixations.

14. Process takes places in 2 step 1 st step-small polymers are formed 2 nd step small polymers cross-link Formations of cross linkages between Aldehyde and protein is measured by changes in viscosity, mechanical strength and molecular size of protein.

15. Formaldehyde Slow reaction Reversible*(in first 24 hr with excess of water) Not good morphological picture Less effective at cross linking Loss of enzyme and immunological activity is less Glutaraldehyde Rapid Irreversible Good morphological picture More effective at cross linking Loss of enzyme and immunological activity more

16. Oxidizing agents React with protein Forms cross-links with proteins Reflected by rapid increase in viscosity After that decrease in viscosity , that phenomenon is known as secondary liquefaction. Osmium tetroxide is more reactive towards protein.

17. Mercuric chloride It reacts with histidine residues in proteins. Also there is production of H+ ions making solution more acidic more efficient. But after fixation ultra structural preservation is poor.

18. Other fixatives Heat fixation /microwave fixation ------ reacts with polar side chains of proteins. This increases their thermal energy which cause denaturation of proteins. This brings about tissue stabilization.

19. Reaction of fixative with nucleic acid Fixation brings about change in physical or chemical state of DNA or RNA at room temperature. Few fixative react with nucleic acid chemically- including mercury and chromium salts. Heating at 45 and 65 degrees with Aldehyde fixatives, there is uncoiling of RNA and DNA respectively.

20. Ethanol, methanol and Carnoy’s fixative are commonly used. DNA is largely collapsed in methanol and ethanol. Presence of salts is known to be essential for the maximum precipitation of nucleic acid from alcohol.

21. Reaction of fixative with lipids Most of lipids are labile. So lost during routine processing. To demonstrate them frozen section or cryostat is used. Aldehyde fixation: Preservation of lipo proteins (fixation of protein counterpart. ) Eg : phospholipids which contain amino group such as phosphotidyl ethanolamine are fixed by aldehyde . 22

22. HgCl ₂ react with highly unsaturated compound which form complex. It also reacts with lipids known as plasmalogen acetal phosphatides . Additives such as tannic acid may be used for demonstration of lipid with light microscopy.

23. Ultrastructural demonstration---post fixation with osmium tetroxide. Cholesterol may be fixed with Digitonin for Ultrastructural demonstration. 24

24. Reaction with Carbohydrates Single fixative not satisfactory. Alcoholic or picric acid fixatives preservation of glycogen which appear coarse eg : Alcoholic formaldehyde, Rossman’s solution. Ultra structural studies gluteraldehyde is satisfactory while potassium permanganate increase image contrast. 25

25. Tanic acid and cetyl pyrimidium have been found useful. Additives to vehicle like Alcian blue or ruthenium red enhance glutaraldehyde fixation of glycogen and mucins. 26

26. Reagents used in fixation Aldehydes : formaldehyde, glutaraldehyde Metallic: mercuric chloride, lead fixative Picric acid fixative Alcoholic fixative Chromate fixative Osmium tetra oxide fixative Acetate fixative

27. Formaldehyde Powerful reducing agent. Most common fixative for routine fixation of biopsy specimen. Formalin: 40%formaldehyde gas in water. Forms methelene bridges between protein molecules. Method 4mm block - 8hrs at room temperature 4mm block - 2hrs at 45°C 28

28. Most commonly is used fixative 10% formalin consist of Formalin (40% formaldehyde) 10 ml Water 90 ml

29. Neutralization is necessary due to formation of formic acid by addition of buffer to maintain pH of 7. Buffer added: phosphate buffer magnesium carbonate Protein groups involved in formation of cross links amino, imino, peptide, hydroxyl, carboxyl and sulphahydryl . Formaldehyde is also obtainable in a stable solid form composed of high molecular weight polymers known as paraformaldehyde .

30. Advantages: Cheap, easy to prepare, relatively stable, staining without preliminary procedures. Good preservation of cell morphology Good penetration properties. Do not cause excessive hardening. Best fixative for nervous system 31

31. Disadvantages- Slow fixation reaction . Morphological details less accurate than glutaraldehyde. Dermatitis of hand. Fumes irritating to nostrils. In tissue containing blood , dark brown artifact pigment granules are formed. 32

32. Fixative Formula Advantages Disadvantages Uses 10%formal saline Water- 900ml Nacl - 8.5gm Formalin-100ml Less shrinkage Even fixation Easy sectioning Good staining Slow fixative Hard tissues Neurological tissues Gross specimen fixation. 10% formalin 40%formaldehyde-100ml Distilled water-900ml Prevents pigments Good fixation Good penetration Preserves Enzymes and organelles Longer time for fixation Routine specimen Used for IHC 10% buffered neutral formalin Formalin -100ml Water - 900ml NaH₂Po ₄-3.5gm Na₂HPo ₄-6.5gm Most routine purpose Stops formation of formalin pigment Fixes tissue rapidly Loss of basophillic staining of the cytoplasm and nucleus Loss of reactivity of myelin to weigert iron haematoxylin method 33

33. Fixative Formula Advantage Disadvantage Calcium acetate formalin (formal calcium) Distilled water- 90ml Calcium acetate monohydrate- 2gm Formalin - 10ml Buffered at pH7 by acetate Preserves phospholipids Less hardening or damage Sectioned easily Artifacts due to calcium Alcoholic formaldehyde Formalin- 100ml 95%alcohol- 900ml Calcium acetate-0.5gm Rapid Fixation Glycogen is better preserved RBC are lysed Formol calcium Formalin- 100ml Distilled water- 900ml 10%calcium chloride-100ml Preservation of lipids Artifacts due to calcium Neutral buffered phenol formalin Neutral buffered formaldehyde-100ml Phenol- 20gm Stops formation of formalin pigment Fixes tissue rapidly 34

34. Glutaraldehyde Introduced by Sabatini, Bensch and Barrett It is a dialdehyde. Stable in acid solution: in pH 3 to 5 at 0 ° to 4° C Used in electron microscopy with osmium tetraoxide . 35

35. Fixation of small tissue: 2.5% solution for 2-4 hrs at room temperature Fixation of large tissue: 4% solution for minimum 6-8hrs fully fixed for 24hrs 36

36. Advantages : Better preservation of cellular and fluid proteins than formaldehyde More stable cross linkages More rapid fixing action than formalin. Less shrinkages than formalin Give better section of blood clot and brain Does not corrode metal More pleasant and less irritating 37

37. Disadvantages More expensive Less stable Penetrates tissue more slowly than formalin Inferior to formalin for PAS t echnique. 38

38. Metallic fixative mercury Mercuric ions act chiefly by combining with the acidic group of proteins and strong combination with sulfur thiol radicles. Advantages: Better staining of nuclei and connective tissue. Give best results with metachromatic staining Routine fixative of choice for preservation of detail of photography. 39

39. Disadvantages Corrode all metal except nickel alloy. Solution deteriorates rapidly. Reduce amount of demonstrable glycogen. Penetration is slow. Long time fixation results in unduly hard and brittle tissue. Diffuse black granules are seen in tissue fixed with HgCl ₂. 40

40. Fixative Formula Type Advantage Disadvantage Use Zenker’s fluid HgCl 2 -50gm K(CrO 4 ) 2 -25gm K 2 SO 4 -10gm Water -1000ml add glacial acetic acid just before use 50 ml MA and cytoplasmic fixative Enhanced C/T Staining Stable for several months Rapid and even penetration Lysis of RBC Removes much iron from haemosiderin Tissue brittleness Blood containing Specimen Eg : spleen Hellys formal zenker HgCl 2 -50gm K(CrO 4 ) 2 -25gm K 2 SO 4 -10gm Water -1000ml add formalin just before use 50 ml MA and cytoplasmic fixative Preferred for blood forming or blood containing eg . Spleen ,bone marrow Irritatational as it contains potassium dichromate and formaldehyde Slower than zenker’s fluid. Blood containing Specimen Eg;spleen,bone marrow 41

41. Fixative Formula Advantage Disadvantage Use Susa fluid HgCl 2 - 45gm NaCl - 5gm Formalin - 200ml Distilled water- 800ml Glacial acetic acid- 40 ml CH 3 COOH- 20gm Minimum shrinkage Rapid and even penetration Excellent staining obtained after its use Direct transfer to 95% alcohol Dissolution of cytoplasmic Granules Intolerant fixative Tissue left long in it are excessively hardened Best for Silver impregn technique. Formal Sublimate HgCl 2 -900ml Formalin -100ml Brilliant staining Enhanced Metachromatic Properties RBC’s are Preserved Direct transfer to 70% alcohol Tissue shrinkage and hardening. Formation of black pigment. Routine post- mortem specimen Satisfactory result with silver impregnation technique for central nervous system 42

42. Fixative Formula Type Use Schaudinns sublimated alcohol HgCl ₂ - 3 gm - Distilled water- 50 ml Absolute alcohol Cytoplasmic fixative Most useful fixative for smears of loose cells on a slide. 43

43. Picric acid fixative It reacts with histone and basic proteins and forms crystalline picrates with amino acid. It preserves glycogen well. Disadvantage: Considerable shrinkage of tissue. It dyes the tissue - yellow colour . 44

44. Fixative Formula Type Advantage Disadvantage Bouin’s fluid 1-2%Picric acid (saturated) -75ml Formalin -25ml Glacial acetic acid – 5ml CH 3 COOH MA Excellent Trichrome Staining( mallory’s , heidenhains , massons aniline) Rapid, even Penetration Shrinkage is slight. Stable solution lyses RBC Reduce the amount of demonstrable iron Lipids are both altered and decreased Tissue crumble when frozen section are cut Not Reliable for cytology Detailed Nuclear Studies Embryo Fixation Good for glycogen Gendre’s fluid Formalin -15ml 95%C 2 H 5 OH saturated with Picric acid -80ml Glacial acetic acid -5ml MA Less shrinkage due to acetic acid. Imparts yellow color Preservation of glycogen and nucleoproteins 45

45. Rossman’s fluid Formalin 10ml C 2 H 5 OH saturated with Picric acid -90ml MA Rapid penetration due to alcohol Imparts Yellow color with shrinkage Carbohydrate fixation Brasil’s alcoholic pure formal fixative Picric acid 1gm 80%Ethanol - 150ml 40% formaldehyde- 60ml Glacial acetic acid-15ml Used by Haust : Formalin- 2040ml Picric acid 80gm Ethanol or isopropyl alcohol- 6000ml CH₃COOH - 65gm MA Best fixative for glycogen 46

46. Alcoholic fixative Mechanism of action: alcohol denatures and precipitate protein, possibly by disrupting hydrogen and other bonds. 47

47. Fixative Type Formula Advantage Disvantage Use Ethanol and methanol Cytological Cytoplasmic fixative Ethyl alcohol and Methyl alcohol Rapid penetration Inflammable Causes Shrinkage and hardens Smears Glycogen Carnoy’s fixative Cytological Nuclear fixative Abs.Alcohol-60ml Chloroform-30ml Glacial acetic acid-10ml Excellent Nuclear fixation and Rapid penetration Destroys Cytoplasmic Elements & lipids Glycogen Preservation 48

48. Fixative Formula Type Advantages Use Clarke’s Fixative Abs.alcohol - 75ml Glacial Acetic acid- 25ml Cytological Nuclear fixative Good penetration and nuclear details preserves cytoplasmic elements Smears and chromosome study. Alcohol formalin 95%ethanol Formalin-10ml Useful fixative for sputum 49

49. Chromate fixative Chromium salts in water form Cr-O-Cr complexes which have affinity for acidic and hydroxyl group of proteins so that complexes between adjacent protein molecules are formed. This leads to disruption of internal salt linkages of protein ,thereby increasing the reactive basic groups and enhancing acidophilia in staining. 50

50. Fixative Formula Type Advantage and use Disadvantage Orth’s fluid 2.5%potassium dichromate-100ml Sodium sulphate-1gm Just before using,formalin-10ml Cytoplasmic fixative Regauds fluid Potassium dichromate Just before use,formalin-20ml Cytoplasmic fixative Demonstration of ,RBC colloid containing tissue, preserve phospholipid Solution darken on standing Prolonged fixation tend to bleach all tissue pigment. Contraindicated in carbohydrates Decrease intensity of PAS reaction. 51

51. Osmium Tetraoxide It is highly reactive substance , being easily reduced. It gels protein probably by a process of bridge formation between compounds. With lipid it forms mono and diester linkages which are then rendered insoluble and non extractable by fat solvent such as alcohol and xylene. 52

52. Osmium tetraoxide Rapid fixing agent Stains tissue structure in a additive way as a grey black deposit.

53. Fixative Formula Type Advantages Disadvantage Use Flemming’s fixative: 1%aquaous chromic acid-15ml 2%aquaous osmiumtetraoxide -4ml Acetic acid -1ml Nuclear fixative It is expensive. Penetration is slow. Difficult to counterstain . Cause reversal of tissue acidophilia In electron microscopy Champy’s fluid 3% Potassium dichromate- 7ml 1% Chromic acid -7ml 2% Osmium tetraoxide - 4ml Cytoplasmic fixative Preserves mitochondria, fat, yolk, lipids Needs to be freshly prepared Preferred for mitochondria 54

54. Effect of fixation   Rule #1 is that fixatives denature macromolecules; Fixation changes the shape of large molecules. This rule is the basis for the varied functions of fixation and why fixed specimens look the way they do under the microscope.  

55. Rule #2 is that different fixatives produce their own morphological patterns. That is an objective fact that does not imply good or bad. Whether we like what we see is a subjective matter predominantly based on our individual training. Many chemicals act as fixatives in that they denature macromolecules, but few produce

56. Rule #3 is that fixation is a chemical reaction that is not instantaneous. Its rate is dependent upon the chemical nature of the fixative solution and its temperature. Freida L. Carson

57. Factors affecting fixation. Hydrogen ion concentration Temperature Penetration Osmolality Concentration duration Other factors: Volume changes Substances added to vehicle 58

58. Hydrogen ion concentration Satisfactory fixation occurs between pH 6 to 8. Stabilization of tertiary and quaternary structure of proteins By addition of acids pH decreases destruction of proteins and cause precipitation. Hence, fixatives must be neutralized by adding buffer. 59

59. Commonly used buffer system are : Phosphate, s- collidine , veronal acetate, Tris and cacodylate . pH chosen must be as near the biochemical optimum as possible. For electron microscopy , tissue must be fixed with a gradually increasing pH 60

60. Temperature High temperature Rapid fixation reactions favors fixation. Fixation should be carried out at gradually increasing temperatures Disadvantages : 1. Risk of tissue distortion 2. Deleterious effect on certain antigen. Use : 1.Rapid fixation of urgent biopsy specimen. 2.To fix tissue with tuberculosis formaldehyde at 100°C is used. 61

61. Low temperature Low temp. Slows down Autolysis more accurate details. Ultra structure and enzyme histochemistry and electron microscopy , temp. range of 0 – 4 degrees is required. 62

62. Penetration Fixation depends on diffusion of fixative into the tissue. Penetration of fixatives is a slow process. Size of specimen is important to ensure complete penetration of fixatives. Small or thin slices of blocks - satisfactory fixation Large blocks of specimen - slow fixation 63

63. Slow rate of diffusion and reaction give rise to various zones of tissue fixed to different degrees. d=k √t (d-depth penetrated , t-time , k-coefficient of infusibility. Fixed tissue acts as a barrier to subsequent inward diffusion of fixatives. 64

64. Osmolality Hypertonic solutions - cell shrinkage. Isotonic and hypotonic solutions - cell swelling In general fixatives that act mainly on protein precipitants cause shrinkage irrespective of what the osmotic pressure is and for non protein precipitants, reverse is true. 65

65. By varying the Osmolality, structure of membrane system within various cells can be altered. Thus , additives to fixatives can alter extracellular space in tissues. Sucrose is commonly added to osmium tetroxide for ultra structural studies Fixative solutions must be preferably isotonic, thus cell swelling is compensated by processing and wax impregnation. 66

66. Concentration Low concentration of fixative with neutral pH favors fixation. Glutaraldehyde solution is used as 3% solution but it is effective even at concentration as low as 0.05% with correct pH of fixative. Presence of buffer causes polymerization of Aldehyde with a consequent decrease in effective concentration. Staining of tissue is altered with the concentration of fixative employed. 67

67. Duration Long duration In Aldehyde : a) inhibit enzyme activity and immunological reactions b) shrinkage and tissue hardening. Glutaraldehyde longer duration of fixation effective polymer formation advantageous. In oxidizing fixatives : degrade the tissue by oxidative cleavage of proteins and loss of peptides. 68

68. i ) Changes in volume- Ideally, changes in processing and fixation cancel each other giving no net change. Formalin fixed tissues along with paraffin embedding causes 33% shrinkage . 69