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FIXATION & FIXATIVES

Dr NAVEEN KUMAR I MDS,OMFP

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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

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GLOSSARY OF TERMSAutolysis – 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.

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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

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INTRODUCTION It is difficult to conduct histochemical investigations upon

living To study the microanatomy For good histological preparations Tissues should be fixed early

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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.”

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.”

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TYPES OF FIXATION Three types of fixation

Heat fixation

Perfusion

Immersion

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Micro anatomical• 10% formol saline• 10% neutral

buffered formalin• Zenkers solution• Bouin’s solution• Rossman’s fluid• Formol calcium

Cytological • 1. Nuclear fixatives • glacial acetic a-

affinity for nuclear chromatin.

• pH≤ 4.6 • Flemming’s• Carnoy’s• Newcomer’s• Clarke’s

• 2. Cytoplasmic fixatives

• glacial acetic a - destroys mitochondria and Golgi.

• pH ≥ 4.6.• Kelly flemmings• Regauds’s fluid• Orth’s fluid

Histochemical • Formol saline 10%• Absolute ethyl

alcohol• Acetone

CLASSIFICATION OF FIXATIVES Based on mode of action

COMPOUND FIXATIVES

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MICROANATOMICAL

Formol calcium (Baker, 1944)Formalin ------------- 10 mgCalcium chloride ---- 2gWater -----------------to 100ml

Buffered formalinFormalin------------------------------- 10mlAcid sod. Phos. Monohydrate--- 0.4gAnhydrous disodium phos. ------ 0.65gWater ---------------------------------to 100ml

Buffered formol sucrose (Holt & Hicks, 1961)Formalin -------------------------10mlSucrose ---------------------------7.5gM/15 phos. Buffer----------to 100ml-preserve- fine structure, phospholipids, enzymes

Acetic – alcoholic- formalin Formalin---------5mlGlacial acetic A----5ml70% alcohol---------90ml-excellent- glycogen-Fix- nuclear protein- rapid, 5mm thick- 4hrs

Formol calcium (Lillie,1965)Formalin...............10mlCalcium acetate....2gWater...................to 100ml

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Zenker’s fluidMer. chlr.-------5gPot. Dichromate---2.5gSod. Sulphate---- 1gDist. Water----- 100mlGlacial acetic A ----- 5ml

-Rapid & even penetration-Fx -12hrs;3mm- 2 to 3 hrsZenker’s formol(Helly’s fluid)Formalin --5ml-Fx – bone marrow, spleen- fx – 6-24hrs

Bouin’s fluidPicric acid sat. aq. Soln-----75mlFormalin(40% formaldehyde)—25mlGlacial acetic acid------------------ 5ml-Penetartes -rapid, even-Brilliant staining – trichome methods-Glycogen-Fx -24hrs-2-3mm thick – 2-3 hrs

Rossman’s fluidFormalin-----10mlAbs. ethyl alcSat. picric acid---90ml- carbohydrates

MICROANATOMICAL

Heidenhain’s susaMercuric chloride....4.5gSod. Chloride..........0.5gTrichloro acetic acid..2gAcetic acid.............4mlFormalin ..............20mlDist. Water..........to 100ml-excellent fx- routine biopsy-brilliant staining; good- cytological detail-penetration- rapid & even-tissues left 24hrs- bleaching, hardening

CARBOHYDRATES

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NUCLEAR FIXATIVES

Carnoy’s fluidAbs. alcohol--- 60mlChloroform---- 30mlGlacial acetic A– 10ml-exclnt nuclear fixation-preserve – nissil substance, glycogen-Very rapid-fx – 1-2 hrs; 2-3mm thick- 15min

Newcomer’s fluidIsopropanol---------60mlPropionic acid-------30mlPetroleum ether-----10mlAcetone----------------10mlDioxane----------------10ml-fx- chromosomes-Fx- 12-18hrs; 3mm thick- 2-3hrs

Flemming’s fluid1% aq. Chromic acid—15ml2%aq. Osmium tetroxide– 4mlGlacial acetic acid ---- 1ml-Poor & uneven penetration-2mm thick- 12hrs

Clarke’s fluidAbs. alcohol------- 75mlGlacial acetic acid--- 25ml-rapid, good nuclear fixation-Excellent- smears

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CYTOPLASMIC FIXATIVES

Champy’s fluid3% pot. Dichromate ------ 7ml1% chromic acid------------7ml2% osmium tetroxide----4ml-Freshly made-Penetration –poor, uneven-Preserves- mito, fat, lipids-2mm thick- 12hrs

Regaud’s fluid3% pot. Dichromate-------80mlFormalin------------------20ml-freshly prepared-Penetrates evenly, rapidly-overharden –tissue-Mitochondia-Fx- 24hrs; 3-4mm thick- 4-6hrs

Muller’s fluidPot. Dichromate------ 2.5gSod. Sulphate---------1gDist. Water-----------to 100ml-rarely used

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Based on chemical agents

1. cross- linking fixatives / aldehydes -formaldehyde, glutaraldehyde2. protein denaturing fixatives/ coagulants - acetic acid, methyl alcohol, ethyl alcohol3. oxidizing agents - osmium tetroxide, potassium permanganate, potassium

dichromate4.Other cross linking agents - carbodiimides5.Miscellaneous - mercuric chloride, picric acid, non-aldehyde –containing

fixatives, dye stuffs

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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

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REACTION OF FIXATIVESReactions with proteins: Maintain morphology- stabilize proteins Cross-links Gel Retain cellular const.

1.Aldehyde

s

2. Oxidiz

ing agent

s3.

Mercuric

chloride

4.Microwav

e

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REACTIONS WITH NUCLEIC ACIDS

Change – physical & chemical -DNA & RNAEg: mercury , chromium salts

1. Aldehydes-Room temp. – x -↑temp. – uncoiling DNA(65◦), RNA (45◦)

2. Alcohols -Commonly used-Removes histone proteins-Extract DNA & RNA

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REACTIONS WITH LIPIDS:

Variable structure, activity – difficult to analyzeConventional hp tech.- lipids removed; cryostats / frozen sections.

1. 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

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REACTIONS WITH CARBOHYDRATES

No single fixative- satisfactory Alcoholic – Glycogen Ultrastuctural studies- tannic acid, acetyl pyridium

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COMMONLY USED FIXATIVESFORMALDEHYDE10% NBF – Common proteins: aqueous solution methylene hydrate (first step) reacts with proteins side chains hydroxy methyl side groups

(characteristic reaction)

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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 reasonablyDoes not cause excessive hardening / brittle

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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

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Chromic acid Potassium dichromate

Picric acid

Dissolving anhydrous chromic trioxide with distilled water

Fixes cytoplasm without precipitation

Explosive- dry,Stored under layer of waterPrecipitates proteins - picrates

advantages

Precipitates- proteinsPreseves- carbohydratesHydrolyses DNA

•Preserves phosphatides- mitochondria

•Gives brilliant contrast•Penetrtion rapid •preserves

disadvantages

Well washed – running water

Well washed – running water

ShrinkageCorrosionCuttingbrownish mercury

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ALCOHOL FIXATIVESAbsolute alcohol

Methanol

ethanol

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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

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Spray fixatives: -alcohol based-aerosol spray cans-fix cell smears on slides-water soluble wax- barrier

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. tetAdvantages Disadvantages -sections- cut easily - extra cost -stain more brilliantly -toxicity- mitochondria

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FACTORS AFFECTING THE QUALITY OF FIXATION

Buffers and pH Penetration (depth ) Duration Temperature Concentration Osmolality Volume Additives

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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

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ARTIFACT PIGMENTSFormalin Mercury Chromic oxide

Colour Brown / blackish

brownish black Yellow- brownRarely 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

Starch – talcum powder - gloves- Maltese cross configuration – PAS +ve

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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

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SUMMARY

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REFERENCES John D Bancroft, Marilyn Gamble – Theory and Practice of

Histological Techniques – 6th edition

A. Culling – Hand book of histopathological and histochemical techniques – 3rd edition

D.J.Cook – Cellular Pathology – 2nd edition

Steven G. Silverberg - Principles and practice of surgical pathology and cytopathology -3rd edition

P. Chakraborty – Practical pathology

Lynch’s medical laboratory technology – S.S.Raphel,W.B.Saunders - 4th 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.