Fixation & fixatives in histopathology, dr naveen reddy

oral pathology

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2. FIXATION & FIXATIVES Dr NAVEEN KUMAR I MDS,OMFP

3. CONTENTS Glossary of terms Introduction Definition Types of fixation Classification of fixatives Effects and aim Reaction of fixatives Commonly used fixatives Factors affecting fixation Fixation for specialized techniques Fixation artefacts summary References 3

4. Glossary of terms Autolysis – lysis or dissolving of cells by enzymatic action, probably as a result of rupture of lysosome . The group of enzymes – cathepsins Putrefaction – breakdown of tissue by bacterial action, often with the formation of gas . Precipitation – a process in which a solid is separated from a suspension or solution . Denaturation  - a major change from the original native state without alteration of the molecule’s primary structure. Osmolality - the molality of an ideal solution of a non dissociating substance that exerts the same osmotic pressure as the solution being considered. 4

5. Additive : they chemically link or bind to the tissue and change it. Non- additive : they act on the tissue without chemically combining with the tissue. Eg : alcohols Coagulant : it will allow solutions to penetrate into the interior of the tissue very easily Non-coagulant : they act by creating a gel like barrier that makes the solution more difficult to penetrate to the interior of the tissue. Birefringence - the double refraction of light in a transparent, molecularly ordered material 5

6. Introduction It is difficult to conduct histochemical investigations upon living To study the microanatomy For good histological preparations Tissues should be fixed early 6

7. Definition Fixative (Dorland’s): “ A fluid, often a mixture of several reactive chemicals , into which histological or cytological specimens are placed so that, by processes such as denaturation and cross-linking of proteins, autolysis is prevented, the specimen is hardened to withstand further processing and the specimen is preserved in a close facsimile of the living state in regard to both cellular morphology and the location of sub cellular constituents.” 7 Fixation:   “ A process by which the constituents of the cells or tissues are fixed in a physical and chemical state so that they will withstand subsequent treatment with various reagents with a minimum loss, distortion or decomposition.”

8. TYPES OF FIXATION Three types of fixation 8 Heat fixation Perfusion Immersion

9. CLASSIFICATION OF FIXATIVES Based on mode of action COMPOUND FIXATIVES 9

10. MICROANATOMICAL Formol calcium (Baker, 1944) Formalin ------------- 10 mg Calcium chloride ---- 2g Water -----------------to 100ml Buffered formalin Formalin------------------------------- 10ml Acid sod. Phos . Monohydrate--- 0.4g Anhydrous disodium phos . ------ 0.65g Water ---------------------------------to 100ml Buffered formol sucrose (Holt & Hicks, 1961) Formalin -------------------------10ml Sucrose ---------------------------7.5g M/15 phos . Buffer----------to 100ml -preserve- fine structure, phospholipids, enzymes Acetic – alcoholic- formalin Formalin---------5ml Glacial acetic A----5ml 70% alcohol---------90ml -excellent- glycogen -Fix- nuclear protein - rapid, 5mm thick- 4hrs Formol calcium (Lillie,1965) Formalin...............10ml Calcium acetate....2g Water...................to 100ml 10

11. Zenker’s fluid Mer. chlr .-------5g Pot. Dichromate---2.5g Sod. Sulphate ---- 1g Dist. Water----- 100ml Glacial acetic A ----- 5ml Rapid & even penetration Fx -12hrs;3mm- 2 to 3 hrs Zenker’s formol ( Helly’s fluid) Formalin --5ml Fx – bone marrow, spleen fx – 6-24hrs Bouin’s fluid Picric acid sat. aq. Soln -----75ml Formalin(40% formaldehyde)—25ml Glacial acetic acid------------------ 5ml Penetartes -rapid, even Brilliant staining – trichome methods Glycogen Fx -24hrs 2-3mm thick – 2-3 hrs Rossman’s fluid Formalin-----10ml Abs. ethyl alc Sat. picric acid---90m l - carbohydrates MICROANATOMICAL Heidenhain’s susa Mercuric chloride....4.5g Sod. Chloride..........0.5g Trichloro acetic acid..2g Acetic acid.............4ml Formalin ..............20ml Dist. Water..........to 100ml - excellent fx - routine biopsy -brilliant staining; good- cytological detail -penetration- rapid & even -tissues left 24hrs- bleaching, hardening CARBOHYDRATES 11

12. NUCLEAR FIXATIVES Carnoy’s fluid Abs. alcohol--- 60ml Chloroform---- 30ml Glacial acetic A– 10ml - exclnt nuclear fixation -preserve – nissil substance, glycogen -Very rapid - fx – 1-2 hrs; 2-3mm thick- 15min Newcomer’s fluid Isopropanol ---------60ml Propionic acid-------30ml Petroleum ether-----10ml Acetone----------------10ml Dioxane ----------------10ml - fx - chromosomes - Fx - 12-18hrs; 3mm thick- 2-3hrs Flemming’s fluid 1% aq. Chromic acid—15ml 2%aq. Osmium tetroxide – 4ml Glacial acetic acid ---- 1ml Poor & uneven penetration 2mm thick- 12hrs Clarke’s fluid Abs. alcohol------- 75ml Glacial acetic acid--- 25ml rapid, good nuclear fixation Excellent- smears 12

13. CYTOPLASMIC FIXATIVES Champy’s fluid 3% pot. Dichromate ------ 7ml 1% chromic acid------------7ml 2% osmium tetroxide ----4ml Freshly made Penetration –poor, uneven Preserves- mito , fat, lipids 2mm thick- 12hrs Regaud’s fluid 3% pot. Dichromate-------80ml Formalin------------------20ml -freshly prepared -Penetrates evenly, rapidly - overharden –tissue Mitochondia Fx - 24hrs; 3-4mm thick- 4-6hrs Muller’s fluid Pot. Dichromate------ 2.5g Sod. Sulphate ---------1g Dist. Water-----------to 100ml -rarely used 13

14. Based on chemical agents 1. cross- linking fixatives / aldehydes -formaldehyde, glutaraldehyde 2. protein denaturing fixatives/ coagulants - acetic acid, methyl alcohol, ethyl alcohol 3. oxidizing agents - osmium tetroxide, potassium permanganate, potassium dichromate 4.Other cross linking agents - carbodiimides 5.Miscellaneous - mercuric chloride, picric acid, non-aldehyde –containing fixatives, dye stuffs 14

15. Effects of fixation Autolysis/ putrefaction Change in shape/ vol. Dessication / shrinkage of tissue Rigidity Penetration Clear staining Tissue- living state Semifluid Semisolid Optical differentiation AIM To preserve tissue To permit 15

16. Reaction of fixatives Reactions with proteins : Maintain morphology- stabilize proteins Cross-links Gel Retain cellular const. 16

17. REACTIONS WITH NUCLEIC ACIDS Change – physical & chemical -DNA & RNA Eg : mercury , chromium salts 17 1. Aldehydes Room temp. – x ↑temp. – uncoiling DNA(65◦), RNA (45◦) 2. Alcohols -Commonly used -Removes histone proteins -Extract DNA & RNA

18. REACTIONS WITH LIPIDS : Variable structure, activity – difficult to analyze Conventional hp tech.- lipids removed; cryostats / frozen sections. 18 Aldehydes >React – phospholipids, unsaturated fatty acids, >Double bond is attacked 2. Mercuric chloride >React -highly unsaturated compounds- complexes > Ultrastructural demo- post fixation – Imidazole Osmium tetroxide >Tannic acid- retain lipids

19. REACTIONS WITH CARBOHYDRATES No single fixative- satisfactory Alcoholic – Glycogen Ultrastuctural studies- tannic acid, acetyl pyridium 19

20. COMMONLY USED FIXATIVES FORMALDEHYDE 10% NBF – Common proteins: aqueous solution methylene hydrate ( first step) reacts with proteins side chains hydroxy methyl side groups ( characteristic reaction) 20

21. Advantages Disadvantages Cheap, easy to prepare, rel. stable May cause dermatitis Allows subsequent appl. Of most staining tech. without spl . Preliminary procedures Fumes might irritate Frozen sections- easily prepared May cause asthma Staining for fat – easily carried out Formation of pigments Penetrates tissues reasonably Does not cause excessive hardening / brittle 21

22. Osmium tetroxide Glutaraldehyde Mercuric chloride Hydrophobic & hydrophilic Nucleic acids* Clumping of DNA- prevented by post fixation KMnO4 / ca ++ , tryptophan during fixation Limited penetration to tissues Secondary fixative – EM Stain lipids – frozen sections Tissue swelling – reversed by dehydration / adding NaCl Black staining Bi functional aldehyde Extensive cross linking for collagen Slow penetration of fixative – any tissue must be small (0.5mm max.) Increased background staining Usage: 4% glut in phosphate buffer at pH7.4 Secondary fixative Reacts – histidine , thiol , phosphate, hydroxyl groups Carboxyl – X Protein precipitant Penetration – rapid & uneven Produces H+ ions- soln more acidic Poor ultra structural preservation 22

23. Chromic acid Potassium dichromate Picric acid Dissolving anhydrous chromic trioxide with distilled water Fixes cytoplasm without precipitation Explosive- dry, Stored under layer of water Precipitates proteins - picrates advantages Precipitates- proteins Preseves - carbohydrates Hydrolyses DNA Preserves phosphatides - mitochondria Gives brilliant contrast Penetrtion rapid preserves disadvantages Well washed – running water Well washed – running water Shrinkage Corrosion Cutting brownish mercury 23

24. 24 ALCOHOL FIXATIVES Absolute alcohol Methanol ethanol

25. Target Fixative of choice Fixative to avoid Proteins NBF, paraformaldehyde Osmium tetroxide Enzymes Frozen sections Chemical fixatives Lipids Frozen sec, glut, osmium tetroxide Alcoholic fixative, NBF Nucleic acids Alcoholic fixatives Aldehydes Muco polysaccharides Frozen sections Chemical Biogenic amines Bouin’s soln , NBF Glycogen Alcoholic fixatives Osmium tetroxide 25

26. Spray fixatives : - alcohol based -aerosol spray cans -fix cell smears on slides -water soluble wax- barrier 26 Vapour fixatives -Fix cryostat cut sections Formaldehyde- heating paraformaldehyde 50C- 80C Acetaldehyde- 80C for 1-4 hrs Glutaraldehyde - 80C for 2min-4hrs POST / SECONDARY FIXATION 2 fixatives in succession Buffered formaldehyde with mercuric chloride For EM, after glut, post fix with Osm . tet Advantages Disadvantages -sections- cut easily - extra cost -stain more brilliantly -toxicity - mitochondria

27. Factors affecting the quality of fixation Buffers and pH Penetration (depth ) Duration Temperature Concentration Osmolality Volume Additives   27

28. Immunohistochemistry -demonstration of antigen- fixative and IHC method -prompt fixation – consistent results Poor fixation – loss of antigenicity / diffusion Eg : 10% NBF, Bouin’s , Formal mercury Enzyme histochemistry -controlled fixation Destroys oxidative enzymes Hydrolytic enzymes – prefixed in cold(4 ◦C) formal calcium Frozen sections -Formalin fixed biopsies- rinsed, -Immersed in 15-20% sucrose for 1-8 hrs at 4 ◦C to replace water before freezing, improve sectioning Electron microscopy Glutaraldehyde conc. Between 1.5% and 4% osmium tetroxide conc. 1% or 2% Fixation for specialized techniques Flow cyometry -1% paraformaldehyde - blood cells -Buffered formaldehyde- fixative of choice 28

29. ARTIFACT PIGMENTS Formalin Mercury Chromic oxide Colour Brown / blackish brownish black Yellow- brown Rarely seen Found blood rich tissues Tissues fixed with mercury containing fixatives Fixed with chr ., dichromate Morphology micro crystalline, bi refringent Extracellular crystal, mono refringent Extracellular and mono refringent Removal 10% ammonium hydroxide in 70% ethyl alcohol Lugol’s iodine and bleaching with weak sodium thiosulfate (hypo) 1% acid alcohol 29 Starch – talcum powder - gloves- Maltese cross configuration – PAS + ve

30. Natural Formalin Substitutes Patil S, Premalatha B R, Rao R S, Ganavi B S. Revelation in the Field of Tissue Preservation – A Preliminary Study on Natural Formalin Substitutes. J Int Oral Health 2013; 5(1):31-38. The possible mechanism of fixation by honey, sugar & jaggery Fructose present in honey, sugar & jaggery Low pH Breakdown to form aldehydes Aldehydes cross-link with tissue amino acids (Similar to the action of formaldehyde) Tissue fixation 30

31. summary 31

32. R eferences John D Bancroft, Marilyn Gamble – Theory and Practice of Histological Techniques – 6 th edition A. Culling – Hand book of histopathological and histochemical techniques – 3 rd edition D.J.Cook – Cellular Pathology – 2 nd edition Steven G. Silverberg - Principles and practice of surgical pathology and cytopathology -3 rd edition P. Chakraborty – Practical pathology Lynch’s medical laboratory technology – S.S.Raphel,W.B.Saunders - 4 th edition Shankargouda Patil , Premalatha B R, Roopa S Rao, Ganavi B S - Revelation in the Field of Tissue Preservation – A Preliminary Study on Natural Formalin Substitutes: J Int Oral Health 2013; 5(1):31-38. 32