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TISSUE PROCESSING :- Tissue processing can be performed either manually or through automated tissue processor. The device can handle larger number of tissues, process more quickly and produces better quality outcome. Two types of devices are available � Tissue transfer or dip dunk � Fluid transfer or enclosed Advantages of automated tissue processor – Saves time, decreases human error, effective fluid circulation, Temperature can be adjusted and vacuum/pressure can also be incorporated. Tissue Transfer Type:-  The machine consist of a time clock, a circular superstructure that contains basket carrier, a receptacle basket and receptacles (stainless steel or plastic capsules), and a circular deck which holds the reagent beakers and plastic baths. Small blocks of tissue are enclosed in the perforated capsules. These capsules are placed in the basket which in turn is attached to one of the yokes in the superstructure, while it is in the raised position. When the superstructure descends the basket is immersed in the first solution and other reagent beakers are covered preventing evaporation of reagents. To move the basket from one reagent to the next the entire superstructure ascends and descends at scheduled intervals controlled by the time clock. During immersion the basket rotates so the infiltration of fluid into the tissues is optimum. The entire process takes about 16 hours. The machine is started in the evening so that the process is complete in the morning,and embedding is done.   Enclosed Type In this type of tissue processor the tissues remain in one container but reagents get changed at scheduled interval. Manual:– In this process the tissue is changed from one container of reagent to another by hand. Advantages:- *Can be used when the number of tissue blocks is limited *Non-availability of automated tissue processor Disadvantages :- *Difficult to use when large number of tissue blocks are to be processed *Proper agitation of reagent not achieved *More evaporation of reagents *Process is tedious and requires constant attention Precautions:- 1. Labels should be written with graphite pencil, India ink or typed. 2. The fluid used in complete dehydration and clearing tend to become contaminated with fluid carried over from previous vat by the tissue. Every alternate day daily the last solution is the series are replaced by fresh solution of 100% alcohol, acetone and xylene and the previously used once one moved forward while the first one is discarded. Other reagents are changed twice a week or earlier with an average work load. It is far better to change the reagents a day earlier than to have a precious surgical specimen improperly infiltrated.  

INTRODUCTION:- The technique of getting fixed tissues into paraffin is called tissue processing. This describes the steps required to take animal and human tissues from fixation to the state where it is completely infiltrated with a suitable wax i.e. paraffin wax and can be embedded and ready for section cutting on microtome OBJECTIVES:- After reading this lesson, you will be able to: *define tissue processing *describe its aim and method of processing. Aim: To process the fixed tissue into a form in which it can be made into thin microscopic sections. Processing:  The steps in this process are dehydration and clearing. Dehydration:  It is the process of removing water from tissues. It is important because paraffin is not miscible with water. Dehydration is usually complete when less then 3-4% of water remains in the tissues. Time required for this depends on: 1. Permeability of tissues 2. Continuous rotation of fluid to prevent stagnation of fluid around tissues 3. Temperature 4. Vacuum applied Dehydrants: Ethyl alcohol, Methyl alcohol, Butyl alcohol and Isopropyl alcohol. The most commonly used dehydrant is ethyl alcohol. Alcohol Method:- The tissues are passed through a series of progressively more concentrated alcohol baths. Concentration of first alcohol bath depends on the fixative and size and type of the tissue, e.g. delicate tissue needs lower concentration of alcohol and smaller interval between two strengths of alcohol.  Usually 70% alcohol is employed as the first solution and100% as the last solution. After about 40 tissues have passed through the first change of alcohol, it is discarded and all the other changes are brought one step lower. Absolute alcohol at the end is always fresh. Usually tissues are kept in each solution for 40 to 60 minutes. Use of copper sulphate in final alcohol:  A layer of anhydrous CuSO4 is placed at the bottom of a dehydrating bottle or beaker and is covered with 2-3 filter paper of approximate size to prevent staining of the tissue. Anhydrous CuSO4 removes water from alcohol as it in turn removes it from tissues. Anhydrous CuSO4 is white in colour while the hydrated form is blue. Therefore, it acts as an indicator for the presence of water. Advantage of CuSO4 1. Rapid dehydration 2. Prolongs life of alcohol 3. Blue colouration of CuSO4 indicates that both alcohol and CuSO4 should be changed Acetone – Acetone is clear colourless inflammable fluid which is miscible with water, ethanol. It is used for complete dehydration. Four changes of acetone of half an hour or two changes of one hour are given to achieve complete dehydration of tissues. Advantages *Rapid actio                                                                                                                                                                                                *Easily removed by most clearing agents                                                                                                                                            *Less expensive Disadvantages:- *Highly volatile *Causes shrinkage and brittleness of tissues *Dissolves lipid more than ethanol Clearing :- Clearing is a process which leaves the tissues clear and transparent. This term relates to the appearance of the tissues after the dehydrating agent has been removed. If the refractive index of the clearing agent is similar to the protein of tissue the tissue becomes transparent. The end point of clearing can be noted by the transparent appearance of the tissue. Thus clearing serves two purposes 1. Removes alcohol to make paraffin impregnation complete 2. Acts as solvent for the mounting media which renders the tissues transparent and improves the refractive index, making microscopic examination easier. Clearing Agents:-  Xylene – It is colourless and most commonly used. Two changes of one hour each are given to get the end point. Prolonged treatment hardens the tissues. It is not preferred for brain tissue. Other Clearing Agents:-  Toluene Dioxane Cedarwood oil Cloroform  Benzene Carbol-xylene – clears rapidly, it is kept reserved for material difficult to clear INFILTRATION AND IMPREGNATION:- After clearing, tissues are transferred to molten paraffin wax for filtration and impregnation. During this process clearing agent diffuses out and molten wax is infiltrated. The wax which has infiltrated in the tissue gets deposited. This process is called impregnation. Routinely two changes are given in the wax to get proper impregnation. The duration and number of changes required for thorough impregnation of tissue depends on  1. Size and type of tissues-Longer time is required for thicker tissues. Vacuum reduces the time required for complete impregnation. 2. Clearing agent employed 3. Use of vacuum imbedding Tissue processing may be performed manually or with the help of automated tissue processor. Routinely 12 containers containing different solutions are used for processing in the following order 10% formalin – container no 1, 2 50% alcohol – container no 3 90% alcohol – container no 4 & 5 Absolute Alcohol – container no 6 Acetone – container no 7 & 8 Xylene – container no 9 & 10 Paraffin Wax – container no 11 & 12      

INTRODUCTION:- It is a process by which the cells or tissues are fixed in chemical and partly physical state so that they can withstand subsequent treatment with various reagents, with minimal distortion of morphology and no decomposition AIMS OF FIXATION:- (a) To preserve the tissues as close to their living state as possible (b) To prevent autolysis and bacterial attack (c) To prevent tissues from changing their shape and size during processing (d) To harden the tissues (e) To allow clear staining of sections subsequently (f) To improve the optical differentiation of cells & tissue PRINCIPLE OF FIXATION:- Fixation results in denaturation and coagulation of protein in the tissues. The fixatives have a property of forming cross links between proteins, thereby forming a gel, keeping everything in their in vivo relation to each other. PROPERTIES OF FIXATIVES AND FACTORS AFFECTING FIXATION:- 1. Coagulation and precipitation of proteins in tissues. 2. Penetration rate differs with different fixatives depending on the molecular weight of the fixative 3. pH of fixatives – Satisfactory fixation occurs between pH 6 and 8. Outside this range, alteration in structure of cell may take place. 4. Temperature – Room temperature is alright for fixation. At high temperature there may be distortion of tissues. 5. Volume changes – Cell volume changes because of the membrane permeability and inhibition of respiration. 6. An ideal fixative should be cheap, nontoxic and non-inflammable. The tissues may be kept in the fixative for a long time TYPE OF FIXATION:- Immersion fixation Perfusion fixation Vapour fixation Coating/Spray fixation Freeze drying Microwave fixation/Stabilization The most commonly used technique is simple immersion of tissues/smears in an excess of fixative. For all practical purposes immersion fixatives are most useful. These may be divided into routine and special SIMPLE FIXATIVES :- 1. Formaldehyde:- Commercially available solution contains 35%-40% gas by weight, called as formalin. Formaldehyde is commonly used as 4% solution, giving 10% formalin for tissue fixation. Formalin is most commonly used fixative. It is cheap, penetrates rapidly and does not over- harden the tissues. The primary action of formalin is to form additive compounds with proteins without precipitation. Formalin brings about fixation by converting the free amine groups to methylene derivatives. If formalin is kept standing for a long time, a large amount of formic acid is formed due to oxidation of formaldehyde and this tends to form artefact which is seen as brown pigment in the tissues. To avoid this buffered formalin is used. 2. Absolute alcohol – it may be used as a fixative as it coagulates protein. Due to its dehydrating property it removes water too fast from the tissues and produces shrinkage of cells and distortion of morphology. It penetrates slowly and over-hardens the tissues 3. Acetone :– Sometimes it is used for the study of enzymes especiallyphosphatases and lipases. Disadvantages are the same as of alcohol. 4. Mercuric chloride :– It is a protein precipitant. However it causes great shrinkage of tissues hence seldom used alone. It gives brown colour to the tissues which needs to be removed by treatment with Iodine during dehydration 5. Potassium dichromate – It has a binding effect on protein similar to that of formalin. Following fixation with Potassium dichromate tissue must be well washed in running water before dehydration. 6. Osmic acid :– It is used for fixation of fatty tissues and nerves. 7. Chromic acid – It precipitates all proteins and preserves carbohydrates. Tissues fixed in chromic acid also require thorough washing with water before dehydration. 8. Osmium tetraoxide – It gives excellent preservation of cellular details, hence used for electron-microscopy. 9. Picric acid – It precipitates proteins and combines with them to form picrates. Owing to its explosive nature when dry; it must be kept under a layer of water. Tissue fixed in picric acid also require thorough washing with water to remove colour. Tissue can not be kept in picric acid more than 24 hrs. COMPOUND FIXATIVES:- 1. Formal saline :– It is most widely used fixative. Tissue can be left in this for long period without excessive hardening or damage. Tissues fixed for a long time occasionally contain a pigment (formalin pigment). This may be removed in sections before staining by treatment with picric alcohol or 10% alcoholic solution of sodium hydroxide. The formation of this pigment can be prevented by neutralizing or buffering the formal saline. Fixation time – 24 hours at room temprature 2. Formal calcium – Useful for demonstration of phospholipids. Fixation time-24 hours at room temperature 3. Zenker’s fluid – It contains mercuric chloride, potassium-di-chromate, sodium sulphate and glacial acetic acid. Disadvantages :-– After fixation the tissue must be washed in running waterto remove excess dichromate. Mercury pigment must be removed with Lugol’s iodine. 4. Zenker’s formal (Helly’s fluid) – In stock Zenker’s fluid, formalin is added instead of acetic acid. Advantages – excellent microanatomical fixative especially for bone marrow, spleen & kidney. 5. Bouins fluid – It contains picric acid, glacial acetic acid and 40% formaldehyde. Advantages – (a) Rapid and even penetration without any shrinkage. (b) Brilliant staining by trichrome method. It is routinely used for preservation of testicular biopsiesntages – even penetration, rapid fixation Points to Remember :- 1. 10% buffered formalin is the commonest fixative. 2. Tissues may be kept in 10% buffered formalin for long duration. 3. Volume of the fixative should be atleast ten times of the volume of the specimen. The specimen should be completely submerged. 4. Special fixatives are used for preserving particular tissues. 5. Formalin vapours cause throat/ eye irritation hence mask/ eye glasses and gloves should be used. 6. Tissues should be well fixed before dehydration. 7. Penetration of fixatives takes some time. It is necessary that the bigger specimen should be given cuts so that the central part does not remain unfixed. 8. Mercury pigment must be removed with Lugol’s iodine. 9. Biopsies cannot be kept for more than 24 hours in bouin’s fluid without changing the alcohol. 10. Glutaraldehyde and osmion tetraoxide are used as fixatives for electron microscopy. Most Commonly used Fixatives in the Laboratory are 10% Formalin.