SEMINAR

TOPIC: FIXATION

SUBMITTED BY RINSEENA M. GAFOOR

SUBMITTED TO Mr. REJO JACOB JOSEPH & Mrs.ARCHANA MADAM

FIXATION

DEFINITION:- It is the process by which the morphology and microscopic anatomy of the tissue is preserve as life like .Fixation prevents autolysis and putrefaction changes in the tissue. Putrefaction is caused by bacterial invasion and subsequent destruction of the tissue.

AUTOLYSIS It is a self destruction due to the action of enzymes which are liberated as soon as cell dies. Autolysis leads to the disappearance of nuclei, cloudiness of the cytoplasm and cell loses its staining property. so these changes in the cell can be prevented either by freezing or by adding some chemical substance called fixative to the tissue .

Most of the fixative act by denaturing or precipitating protein which will form a network , which make all other cell components

Aim And Functions of Fixative Fixative should prevent autolysis and putrefaction by rapid coagulation of the cytoplasm. Minimize the risk of infection. It should not shrink or swell, nor destroy the structure of the tissue. It should rapidly and evenly penetrate the tissue. It will provide a condition to allow clear staining of section. It will not alter any chemical changes of the cell. To keep the tissue to the living state without loss of rearrangement.

FACTORS INVOLVING FIXATION pH and Buffer Generally satisfactory fixation occurs at the pH 6.8. For some purpose fixative at specific Ph is chosen e.g.: For the fixation of gastric mucosa, pH is 5.5. Acetic acid is used for lowering the pH Few buffers can also be used e.g.: phosphate buffer, bicarbonate buffer

Temperature Lower temperature slows down autolysis and higher temperature enhances the rapidity of fixation Routinely fixation carried out by room temperature For electro microscopy and histoscopy temperature range chosen is 0.4 c. Formalin heated at 60 c is used to fix the urgent biopsy specimen, but higher temperature may destroy the cells or tissue..

Penetration Depth of the penetration D α √t D=k √t d=mm t=hrs k=diffusion coefficient

Osmolarity Best results are obtained by using slightly hypertonic solution is used. Concentration It is determined by solubility, staining pattern and its effectiveness. 10% formalin is used routinely for brain fixation. 15% formalin is used for friezing the time of 3 % gluteraldehyde. Is used for electron microscopy.

Duration Prolong fixation in formaldehyde will cause shrinkage and hardening and will cause destruction of some enzyme, but in gluteraldehyde prolong fixation is advantage than formaldehyde.

THEORETICAL ASPECTS OF FIXATION Reaction of fixative with protein Most of the important reaction during fixation is to stabilize proteins Aldehydes It cause cross links with protein. The reaction is pH dependent _ i.e. reaction is more rapid at high pH. Reaction of formaldehyde is reversible with in 24 hrs but with neutraldehyde it is rapid and irreversible.

Oxidizing Agents Osmium tetroxide form cross links with proteins where as potassium permanganate and potassium dichromate unless reactive towards protein.

Non chemical fixative Heat and microwave fixative are non-chemical type of fixative. Direct flaming leads to the destruction of tissue. Using microwave fixative heating is controlled. Using microwave fixative tissue molecules can be oscillated and high frequency leads to generate internal heating which fixed the protein by denaturation. it is very rapid method and it is used when rapid diagnosis is needed

Reaction of fixation with lipids Fixative used for lipid demonstration is aldehyde. Following aldehyde fixation, most lipids are not stable but it removes during conventional histopathological process. Reaction of fixative with carbohydrate 60_80% glycogen will lose in aqueous solution, so alcoholic fixatives are recommended for glycogen fixation. .

Reaction of fixative with nucleic acid Ethanol, methanol, & carnoy’s fixative are used for demonstration of nucleic acid. These fixatives bring about the physical and chemical changes of nucleic acid

CLASSIFICATION OF FIXATIVE Fixatives are classified into SIMPLE and COMPOUND Based on chemical action fixatives are further classified into ALDEHYDE Formaldehyde Gluteraldehyde METALLIC FIXATIVE Mercuric chloride Lead fixative OXIDIZING AGENTS Osmium tetroxide K+ permanganate K+ dichromate PROTIEN DENATURING AGENTS Acetic acid Methyl alcohol Ethyl alcohol MISCELANEOUS Picric acid Non aldehyde

FORMALDEHYDE Formalin is a routine fixative. Commercially available formaldehyde is called formalin. 40% formaldehyde is considered as 100% formalin. This is a saturation of formaldehyde gas. 10% formalin_10ml dissolved in90ml of water. Action Formaldehyde reacts with proteins to form cross links between the molecules and form insoluble product. b) Formaldehyde reacts with lipids cause degradation. c) Formalin becomes cloudy in all atmospheres or in long storage due to formation of Para formaldehyde. It is prevented by adding methanol. d) Formalin is usually acid due to the formation of acid content which may cause the formalin

Advantage It is cheap and easy to prepare. It penetrates rapidly and never hardens the tissue. Natural color can be restored, so it can be used in museum mounting. It allows most of the staining property without any chemical changes. Disadvantages It will cause irritation to nose, eyes, and skin. Hence adequate ventilation and the use of gloves are essential. It is not suitable in electron microscopy. It forms white precipitate or Para formaldehyde to form black color formalin pigment due to the formic acid and will cause misinterpretation.

Gluteraldehyde It is mainly used for electron microscopy and usually used in combination with osmium tetroxide. It has more rapid action than formaldehyde. Disadvantage It is expensive. Its penetration is poor. It over hardens the tissue.

It is a protein precipitate which rapidly penetrates and hardens the tissue. Disadvantage Formation of black or brown color pigment. This can be removed by treatment of tissue in 5% iodine prepared in 80% alcohol. Keep for 5 min. Then rinse in running tap water. Keep in aqueous sodiumthiosulphate for 30min. Then again wash in running tap water. Mercuric chloride

Osmiumtetroxide It demonstrates lipids. It gives excellent preservation of cells. So it is used in electron microscopy. Disadvantage It is expensive. It penetrates poorly. It is a very irritating chemical and will cause conjunctivitis. It will be very easily reduced and it should be stored in dark place. It react lipids and form blackening.

Potassium dichromate It has binding effect on protein, similar to that of formalin. It gives fixation of cytoplasm without precipitation of proteins. It preserves phosphotides and it is used for the fixation for the fixation of mitochondria. Following fixation in potassium dichromate, tissue must be washed in running tap water before dehydration, otherwise will cause formation of insoluble lower oxide.

Chromic Acid It is prepared by dissolving anhydrous chromium tetroxide in distilled water. It precipitates protein and preserve carbohydrate. It is a powerful oxidizing agent. Acetone It is used for the histochemical demonstration of tissues and enzymes. Disadvantage Glycogen is not preserved by using acetone.

Picric Acid It precipitates protein. It gives a brilliant contrast for trichrome stain. Disadvantage Formation of insoluble picrate. It is explosive, causes lyses of red cells and remove iron from the tissue. Ethyl alcohol It denatures protein and precipitates them. It also precipitates glycogen. It is used for glycogen demonstration. When it is fixed with other reagents such as carnoy’s, fixation is rapid. It dissolves fats and lipids. It penetrates slowly and makes to harden the tissue.

Acetic Acid It is not usually using alone as it will cause swelling. Trichloro acetic acid It is used for compound fixative. It is a protein precipitates and has a slight decalcifying property.

COMPOUND FIXATIVES These are the product of two or more simple fixative mixed together to get combined effect of their properties. It is classified into three groups. Micro Anatomical Fixatives Cytological Fixatives Histochemical Fixatives

MICROANATOMICAL FIXATIVES It is used to preserve the anatomy of the tissue with correct relationship of tissue layers and large aggregates of cell. Formal calcium Formalin- 10ml Calcium chloride- 2gm Water- 90ml Advantage It is used for the fixation of lipids. It prevents the formation of formalin pigment Formal saline Nacl- 9gm, Formalin pigment will form. It is ideal for fixing the brain.

Buffered formalin Formalin – 10ml NaH2Po4 – 0.4gm Na2HPo4 – 0.65gm Water – 90ml Advantage Formalin pigment will not form. Sucrose – 7.5gm Phosphate buffer – 100ml s

Advantage This fixative gives excellent preservation of fine structure, phospholipids and some enzymes. It is recommended for cytochemistry and electron microscopical studies. For best results it is used at 4 Mitochondria and endoplasmic reticulum is well preserved in electron microscopical pictures by these fixatives.

Alcoholic formalin Formalin – 10ml 95% formalin – 90ml Calcium acetate – 0.5gm Advantage Rapid fixation and it is a good glycogen fixative Disadvantage It will cause shrinkage Acetic Alcoholic Formalin Formalin – 5ml Glacial acetic acid – 5ml 70% alcohol – 90ml Excellent glycogen preservation Rapid fixation It is not ideal for routine fixative

Buffered gluteraldehyde Gluteraldehyde stock solution – 16ml Phosphate buffer – 84ml Mercuric Chloride Containing Fixatives Tissue fixed with mercuric chloride containing fixative cause the formation of black precipitate of mercuric pigment. This pigment has to remove from the deparaffinised section before staining. This is done by treating the section in 0.5% iodine and prepared in 80% alcohol for 5-10min and washed in running water and decolorize with sodium thiosulphate and after that wash again in running tap water.

Heidenhan’s susa Mercuric chloride – 4.5gm Nacl -0.5gm TCA – 2gm GAA – 4ml Formalin – 20ml Distilled water – 80ml Advantage It is excellent fixative for routine biopsy It gives brilliant staining with good cytological details. Rapid penetration with minimum shrinkage. It is having slight decalcifying power because it is incorporated with TCA. Disadvantages Formation of mercuric pigment and it should be removed by treating with iodine.

Zenker’s Fluid Mercuric chloride- 5gm Potassium dichromate- 2.5gm Sodium sulphate – 1gm Distilled water – 100ml Add glacial acetic acid immediately better use – 5ml Advantages Good routine fixative and excellent staining for connective tissue fibers. Disadvantages Its penetration is poor. After fixation wash the fixed tissue in running tap water to remove excess dichromate

Helly’s fluid Mercuric Chloride – 5gm Potassium dichromate – 2.5gm Sodium sulphate – 1gm Distilled water – 100ml Add formalin before use – 5ml Advantage It is an excellent micro anatomical fixative. It is a very good fixative for bone marrow, spleen and blood containing tissue. Disadvantages It is slow in action than zenker fluid.

ADVANTAGE: IT PENETRATE RAPIDLY IT IS USED FOR THE PRESERVATION OF CHROMOSOME. IT PRESERVE CHROMATIN BETTER THAN CARNOY’S FLUID IT ALSO PRESERVE POLYSACCHARIDE

FLEMINGS FLUID 1% aq CHROMIC ACID - 15ml 1% OSMIUM TETROXIDE -4ml GLACIAL ACETIC ACID – 1ml FOLLOWING FIXATION BY USING TH FLEMMING’S FLUID TISSUE SHOULD BE WASHEDOVER NIGHT. IT IS RARELY USED BECAUSE OF POOR PENERATION FLEMINGS FLUID 1% aq CHROMIC ACID - 15ml 1% OSMIUM TETROXIDE -4ml GLACIAL ACETIC ACID – 1ml FOLLOWING FIXATION BY USING TH FLEMMING’S FLUID TISSUE SHOULD BE WASHEDOVER NIGHT. IT IS RARELY USED BECAUSE OF POOR PENERATION

CYTOPLASMIC FIXATIVE CHAMPY’S FLUID REGAURD’S FLUID SCHAUDINN’S FLUID HELLY’S FLUID MULLER’S FLUID FORMAL SALINE

CHAMPY’S FLUID IT PREVNTS MITOCHONDRIUM, FATS AND LIPIDS IT SHOULD PREPARE FRESHLY BEFORE USE IT PENETRATES POORLY THIN PIECES OF TISSUES HAVE TO BE TREATED FOR THESE FIXATIVES

REGAURD’S FLUID IT SHOULD PREPARE FRESHLY BEFORE USE RAPID PENETRATION IT WILL CAUSE OVER HARDENING OF THE TISSUE IT CAN BE USED AS ROUTINE FIXATIVE IT PRESERVE MITOCHONDRIA VERY WELL

SCHAUDINN’S FLUID IT IS A VERY GOOD CYTOPLASMIC FIXATIVE IT WILL CAUSE EXCESSIVE SHRINKAGE IT IS NOT RECCOMMENDED FOR TISSUE BIOPSY TO REMOVE THE MERCURIC PIGMENT TISSUES OR SMEARS SHOULD BE TREATD WITH IODINE, ALCOHOL AND SODIUM THIOSULPATE WASH IN RUNNIG TAP WATER

HELLY’S FLUID (ZENKER’S-FORMAL) IT CAN BE USED AS A CYTOPLASMIC FIXATIVE PARTICULARLY FOR BONE MARROW AND BLOOD FORMING ORGANS

MULLER’S FLUID IT’S A VERY RARELY USED FIXATIVE

FORMAL SALINE AFTER THE FIXATION USING THE POST CHROMIUM GIVES GOOD CYTOPLASMIC FIXATION.

HISTOCHEMICAL FIXATIVS After using histochemical fixatives cryostat and frozen’s are used for cutting of the section A good histochemical fixative should preserve the constituent to be demonstrated, preferably preserving its morphological relationship Bind or preserve a specific tissue constituent without effecting the relative groups to be used in its visualization.

BUFFERED FORMALIN ADVANTAGE Formalin pigment is not formed by using this fixative’s.

FORMAL SALINE ADVANTAGE Formalin pigment will not form.

COLD ACETONE These fixatives is used in 0-4oc. Its used for the study of enzymes particularly for the phosphates.

ABSOLUTE ALCOHOL 95 % alcohol is called absolute alcohol. It needed 24 hrs fixation. Its occasionally recommended as a basic fixatives , but in most histochemical techniques formalin can be used as an alternative with a consequent improvement in micro anatomical and cytological preservation.

VAPOUR FIXATION Vapour fixatives may be used to fix cryostat –cut section of fresh tissues and sections or blocks of frozen dried tissue. Formaldehyde vapour generated from heated paraformaldehyde is a very high reactivity. Cryostat sections mounted on slides may be placed in a closed vessel above the formaldehyde and the vessel placed in an oven at 60-70˚c for 2 hrs. Using this method we have produced sections showing excellent preservation of glycogen with a very good morphological details

Fixatives which have been used for vapour fixation usually at a temperature of 60- 80˚c. Formaldehyde - 60-70˚c. Acetaldehyde 80˚c-1-4hrs. Gluteraldehyde 80˚c. Osmium tetroxide Diacetyl Acetic acid Glycoxylic acid Glyoxal