Fixation of tissues

Fixation of tissues
Histopathology

1. FIXATION OF TISSUES SUNIL KUMAR.P Haematology & Transfusion Medicine St.John’s Medical College Bangalore. 2/6/2018 1 SUNIL KUMAR.P

2. Definition Aims of Fixation Ideal Characteristics of fixatives Types of fixatives Mode of action Preparation of fixatives and indications Factors affecting fixation 2/6/2018 2 SUNIL KUMAR.P

3. Introduction As soon as cells or tissues are removed from the body they begin to die and undergo post-mortem changes. These changes may be autolytic or putrefactive. 2/6/2018 3 SUNIL KUMAR.P

4. Autolysis : Is a self destructive process due to the release of autolytic enzymes from the dead cells . Putrefaction : Occurs due to the action of bacteria that invade the tissue. 2/6/2018 4 SUNIL KUMAR.P

5. Definition of Fixative A fixative may be defined as a substance which prevent post mortem changes and preserves the morphological and chemical characteristics of cells and tissues. 2/6/2018 5 SUNIL KUMAR.P

6. Aims of Fixation 1.It should prevent autolysis & putrefaction of the cell. 2. It should penetrate evenly and rapidly. 3. It should harden the tissues 4. Increase the optical differentiation of cells & tissues 5. Should not cause shrinkage or swelling of the cells 6. Must not react with the receptor sites & thus must notinterefere with the staining procedure. 7. It must be cheap and easily available. 2/6/2018 6 SUNIL KUMAR.P

7. Good fixative is most important factors in the production of satisfactory results in histopathology. Following factors are important: Fresh tissue Proper penetration of tissue by fixatives Correct choice of fixatives 2/6/2018 7 SUNIL KUMAR.P

8. No fixative will penetrate a piece of tissue thicker than 1 cm. For dealing with specimen thicker than this, following methods are recommended: 1. Solid organ: Cut slices as necessary as but not thicker than 5 mm. 2/6/2018 8 SUNIL KUMAR.P

9. 2.Hollow organ: Either open or fill with fixative or pack lightly with wool soaked in fixative. 3.Large specimen: It requires dissection, Inject fixative along the vessels or bronchi as in case of lung so that it reaches all parts of the organs. 2/6/2018 9 SUNIL KUMAR.P

10. Properties of an Ideal Fixative Prevents autolysis and bacterial decomposition. Preserves tissue in their natural state and fix all components. Make the cellular components insoluble to reagent used in tissue processing. Preserves tissue volume. 2/6/2018 10 SUNIL KUMAR.P

11. Avoid excessive hardness of tissue. Allows enhanced staining of tissue. Should be non-toxic and non-allergic for user. Should not be very expensive. 2/6/2018 11 SUNIL KUMAR.P

12. Mechanism / Action of Fixatives Most fixatives act by denaturing or precipitating proteins which then form a sponge or meshwork, tending to hold the other constituents 2/6/2018 12 SUNIL KUMAR.P

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14. Mechanism / Action of Fixatives At the molecular level, fixative have the property of coagulating proteins in the tissue, through the formation of crosslink's between protein molecules thereby keeping their relation to each other. 2/6/2018 14 SUNIL KUMAR.P

15. Methods of fixation Heat fixation Perfusion Immersion Vapour method Phase partition method 2/6/2018 15 SUNIL KUMAR.P

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17. Classification o f Fixatives 1)Physical Method of fixation. 2)Chemical method of fixation. 2/6/2018 17 SUNIL KUMAR.P

18. Physical method of fixation Heat fixation : The simplest form of fixation is heat. Microwave Fixation: Microwave heating speeds fixation and can reduce times for fixation of some gross specimens and histological sections from more than 12 hrs to less than 20 min. 2/6/2018 SUNIL KUMAR.P 18

19. Freeze-Drying and freeze substitution : Freeze-Drying is a useful technique for studying soluble materials and small molecules; tissues are cut into thin sections, immersed in liquid nitrogen, and the water is removed in a vaccum chamber at -40oc . The tissue can be post-fixed with formaldehyde. 2/6/2018 SUNIL KUMAR.P 19

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21. Chemical Fixation Chemical fixation utilizes organic or non-organic solutions to maintain adequate morphological preservation. Chemical fixatives can be considered as members of three major categories.. 1.coagulant 2.Cross-linking 3.Compound fixatives 2/6/2018 SUNIL KUMAR.P 21

22. Simple Fixatives Formalin The most commonly used fixative is Formalin . It is prepared by mixing 40 % Formaldehyde gas in 100 w/v of distilled water. The resultant mixture is 100 % Formalin. Routinely, 10 % formalin is used which is prepared by mixing 10 ml of 100 % formalin in 90 ml of distilled water. 2/6/2018 22 SUNIL KUMAR.P

23. MECHANISM OF ACTION It forms cross links between amino acids of proteins thereby making them insoluble. It fixes 4 mm thick tissue in 8 hours . 2/6/2018 23 SUNIL KUMAR.P

24. ADVANTAGES : 1.Rapid penetration 2. Easy availability & Relatively cheap(Low cost) 3. Does not over harden the tissue 4. Fixes lipids for frozen sections 5.It is relatively easy to prepare 6.It allows subsequent use of most staining procedures. 7.frozen sections can be made with formalin fixed tissue. 2/6/2018 24 SUNIL KUMAR.P

25. DISADVANTAGES: 1.Irritant to the nose, the eyes and mucous membranes 2. Formation of precipitate of paraformaldehyde which can be prevented by adding 11- 16 % methanol. 3. Formation of black formalin pigment , Acid formaldehyde hematin. 4.It causes shrinkage of collagen. 5.it suspected to contain cancer producing agents 2/6/2018 25 SUNIL KUMAR.P

26. Other Simple Fixatives Glutaraldehyde Osmium Tetraoxide Pottasium Dichromate Mercuric Chloride Picric acid Zenker's fluid Zenker’s Formal (Helly’s Fluid) Bouin’s Fluid 2/6/2018 26 SUNIL KUMAR.P

27. Compound Fixatives Microanatomical fixatives: These are used to preserve the anatomy of the tissue. Cytological fixatives: These are used to fix intracellular structures. Histochemical fixatives : These are used to demonstrate the chemical constituents of the cell 2/6/2018 27 SUNIL KUMAR.P

28. Microanatomical Fixatives 10 % Formal saline : It is a microanatomical fixative. Ideal for fixation of brain. Buffered formalin: Due to the presence of buffer, the pH of the solution remains at neutral or near neutral. As a result, Formalin pigment formation doesn’t take place 2/6/2018 28 SUNIL KUMAR.P

29. Cytological Fixatives Nuclear fixatives : Carnoy’s Fluid Clarke’s Fluid Newcomer’s Fluid Flemming’s Fluid Cytoplasmic Fixatives : Champy’s Fluid Regaud’s Fluid 2/6/2018 29 SUNIL KUMAR.P

30. Special Fixatives Dichromate and chromic acid fixatives 2/6/2018 SUNIL KUMAR.P 30

31. Histochemical Fixatives: Formal saline Cold acetone Absolute alcohol 2/6/2018 31 SUNIL KUMAR.P

32. Composition & Preparation of Fixatives:- 1- 10%Formalin Solution : It is recommended for fixation of general surgical biopsy specimen and tissues from CNS. It causes even fixation and very little shrinkage because of its isotonicity. Composition & Preparation Formaldehyde (37-40%) - 10 ml Distilled water - 90 ml Mix well. 2/6/2018 32 SUNIL KUMAR.P

33. 10% Neutral Buffered Formalin Solution: It is recommended for research specimens but may also be used for surgical and post mortem specimens. The special advantage of NBF is that it prevent the formation of troublesome acid formalin pigment . Disadvantage : Laborious and time consuming Composition & Preparation Formaldehyde (37-40%) - 100 ml Distilled water - 900 ml NaH2PO4 - 4.0 g Na2HPO4 (anhydrous) - 6.5 g Mix to dissolve. 2/6/2018 33 SUNIL KUMAR.P

34. Zenker's Solution( Mercuric Chloride Fixative) M.C. Fixatives rapidly penetrates tissues and permit excellent staining of nuclei and connective tissues. Disadvantage : causes hardening of tissues Composition & Preparation : fixation time 4-24 hours. Distilled water - 950ml Potassium dichromate - 25g Mercuric chloride - 50g Glacial acetic acid - 50g Fixed tissue should be washed overnight in running tap water before processing. 2/6/2018 34 SUNIL KUMAR.P

35. Bouin's fluid fixation time 6 hours. Composition & preparation Saturated aqueous solution of picric acid - 75ml Formalin (~ 40% aqueous solution of formaldehyde) - 25ml Glacial acetic acid - 5ml Fixed tissue should be transferred to 70% alcohol. 2/6/2018 35 SUNIL KUMAR.P

36. Carnoy's fluid fixation time 1-3 hours. Composition & Preparation: Ethanol - 60ml Chloroform - 30ml Glacial acetic acid - 10ml Fixed tissue should be processed immediately or transferred to80% alcohol. 2/6/2018 36 SUNIL KUMAR.P

37. Formal Zenker's(Helly's fluid) By adding formalin to Zenker’s solutions the beneficial effects of both fixatives are combined, minimising their disadvantages. Staining of nucleus, cytoplasm and connective tissue is good with Helly’s fluid. fixation time 12-24 hours. 2/6/2018 37 SUNIL KUMAR.P

38. Formal Zenker's(Helly's fluid) Composition & Preparation: Potassium dichromate - 25g Mercuric chloride - 50g Sodium sulphate - 10g Distilled water - 1000ml Stock solution - 100ml Formalin (~ 40% aqueous solution of formaldehyde) - 5ml 2/6/2018 SUNIL KUMAR.P 38

39. Factors affecting fixation:- 1 – Temperature 2 – Size of the specimen 3 - Volume ratio 4 – Duration of Fixation 5 - Choice of fixatives 6 – Penetration 7- Tissue Storage 8 –Buffer & pH 9 - Osmolality 2/6/2018 39 SUNIL KUMAR.P

40. Fixatives used for Electron Microscopy Glutaraldehyde Osmium tetroxide Formaldehyde & Glutaraldehyde mixture Acrolein 2/6/2018 SUNIL KUMAR.P 40

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