Tissue processing

tissue processing done after the grossing. covers fixation dehydration bluing staining.

1. [email protected] TISSUE PROCESSING

2. CONTENTS Introduction Factors influencing Processing Stages of processing -Fixation -Dehydration -Clearing -Impregnation -Embedding -Sectioning -Staining -Mounting Automated Tissue Processor Bibliography

3. INTRODUCTION Proper handling of tissue specimens is critical to ensure that an accurate diagnosis is obtained from patient tissue samples. Regardless of the methodology, tissue samples requiring processing need to be placed in fixative as soon as possible after excision from the patient. This is essential to prevent autolysis, which could destroy diagnostic elements, and to prepare the tissue for the rigors of the reagents used in subsequent processing steps.

4. Definition: “Tissue processing” describes the steps required to take animal or human tissue from fixation to the state where it is completely infiltrated with a suitable histological wax and can be embedded ready for section cutting on the microtome. Aim: The aim of tissue processing is to embed the tissue in a solid medium firm enough to support the tissue and give it sufficient rigidity to enable thin sections to be cut , and yet soft enough not to damage the knife or tissue.

5. FACTORS INFLUENCING PROCESSING 1. Viscosity : soultions with low viscosity smaller sized molecules faster penetration rate and vice-versa. Melted paraffin wax has a low viscosity which enhances the impregnation rate. 2. Agitation : increases the flow of solutions around the tissue. The mechanism for agitation in automated processors: either vertical/rotary oscillation or pressurized removal and replacement of fluids at timed intervals.

6. 3. Heat : increase in temperature improves the fluid exchange and penetration rate. Excessive exposure to heat can cause shrinkage and hardening of the tissue which negatively affects subsequent staining and immunohistochemistry . 4. Vacuum & Pressure : increase fluid mobility, thus increasing the infiltration rate and decreasing the time necessary to complete each processing step. Vacuum aids the removal of air pockets in porous tissue, e.g. lung.

7. 5. Processing solvent contamination : The number of blocks on each run, tissue type, size, frequency of the runs, use of sponges and cross-contamination of processor solvents will influence how often solutions should be rotated between stations or changed to maintain processing quality. 6. Size : Tissue slice below 4mm are satisfactory.

8. PROTOCOL Fixation Mounting

9. FIXATION The first and most critical step in specimen handling. Fixation denatures proteins rendering the cell and its components resistant to further autolysis. Complete fixation also allows the tissue to withstand the negative effects of subsequent processing reagents. Although samples are typically received in fixative, it is best to begin processing with a fixative station as the natural diffusion of water from the tissue will have diluted the fixative in the specimen container reducing the fixative’s efficacy.

10. The size and type of specimen in the tissue cassette determines the time needed for complete fixation and processing to occur. The tissue should be dissected to 2–4 mm in thickness and trimmed to a size that allows complete flow of reagents around the tissue in the cassette. Ideally tissue should be separated according to size and/or type, and processed using different schedules. The most commonly used reagent for the fixation of histological specimens is 10% neutral buffered formalin (NBF).

11. Post-fixation treatment Specimens fixed in alcoholic fixatives should be followed with alcohol to prevent re-introduction of water to the tissue specimen. Picric acid fixatives will color the tissue bright yellow. A rinse of the tissue in 50-70% alcohol for 4-6 hours will remove excess fixative. This can also be accomplished by treating the cut tissue section on the glass slide with a dilute carbonate solution. A few drops of 1% eosin can be added to the specimen container 30 minutes prior to processing to assist in visualizing small tissue fragments during embedding. The pink color of the tissue remains during processing, but washes out during subsequent staining. Incomplete removal of the eosin can interfere with fluorescent procedures.

12. DEHYDRATION Dehydration displaces the residual fixative as well as cellular water. Correct dehydration schedules during tissue processing should only remove the free water, leaving the bound water intact. Graded alcohols are used in dehydration to remove free water and keep the bound water in place. The removal of the bound water will produce overprocessing artifacts such as shrinkage, ‘parched earth’ effect and abnormal staining, as well as dry, brittle tissues during microtomy .

13. Incomplete dehydration will impair the penetration of the clearing reagents into the tissue, leaving the specimen soft and non-receptive to paraffin wax infiltration. Ethanol, reagent alcohol and isopropanol work well in tissue processing. Glycol ether dehydrants are an effective alternative to alcohols in tissue processing. Due to their gentle nature they are used undiluted.

14. DEHYDRATING AGENTS Alcohol- expensive; dehydrates quickly. Ethyl Alcohol- very expensive Isopropyl Alcohol- used in routine, easily available and cheap. Methyl Alcohol- unpleasant odour ; poisonous. Acetone- causes excess shrinkage and hardening; can be used for small biopsies; dehydrates quickly. Dioxane - dehydrating as well as clearing agent, toxic and carcinogenic.

15. CLEARING/DEALCOHOLIZATION Clearing agents must be miscible with both the preceding anhydrous alcohols/ dehydrants to effectively remove them, and with the ensuing paraffin wax to allow complete infiltration. A good clearant will also dissolve lipids which can impede the wax penetration. This is an intermediate stage between dehydration and Infiltration.

16. Clearing should have following properties: Rapid penetration of tissues Rapid removal of dehydrating agent Ease of removal by melted paraffn wax Minimal tissue damage Low flammability Low toxicity Low cost Clearing agents suitable for routine use: Xylene : Highly inflammable, rarely used. Over exposure causes hardness of tissue. Quick in action, satisfactory.

17. 2. Toluene : Similar to xylene , less damaging than xylene in case of prolonged exposure but it is more inflammable and volatile. Suitable for automated processing. 3. Chloroform : Slower in action, can be used for blocks thicker than 1 mm. It produces highly toxic gases. It is most commonly used in CNS specimens. Requires longer time (overnight) for action. 4. Benzene : Similar to xylene , not used as it is carcinogenic.

18. 5. Petrol : Not used because of various additives. 6. Cedar wood oil : Good clearing agent but slow in action. Expensive; used for research purposes. 7. Clove oil : Same as cedar oil.

19. How is it done?

20. WAX INFILTRATION/ IMPREGNATION The various waxes which are used are: 1. Paraffin wax 2. Resin 3. Agar 4. Gelatin 5. Celloidin 6. Low viscosity nitrocellulose(LVN)

21. BLOCK FORMATION/EMBEDDING It is done by transferring the tissue which has been cleared off the alcohol to a mould filled with molten wax & is allowed to cool and solidify. After solidification, a wax block is obtained which is then sectioned to obtain ribbons. Types of Moulds: A. Leuckhart’s Moulds B. Glass or Metal petri -dishes C. Watch glass D. Paper boats .

22. Embedding is the process by which tissues are surrounded by a medium such as agar, gelatin, or wax which when solidified will provide sufficient external support during sectioning. Paraffin wax It is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils. It is about two thirds the density and slightly more elastic than dried protein. Paraffin wax is traditionally marketed by its melting points which range from 39°C to 68°C. The properties of paraffin wax are improved for histological purposes by the inclusion of substances added alone or in combination to the wax: blended with ceremic , bees wax, rubber etc. This improves ribbonning , increases hardness, decrease melting point, improve adhesion between specimen and wax.

23. OTHER WAXES: Ester wax: M.P- 46-48 C; used in Base sledge microtome. Resins: for electron microscopy. Agar: can be used in double embedding with paraffin wax for friable tissue. Gelatin: used when frozen section of friable tissue is required; also useful in uterine curettings . Celloidin : not used in routine; useful in neurological specimens; supplied in the form of wool dampened with alcohol; working strength- 2 to 4% LVN: used in preference to celloidin since it forms harder blocks; thinner sections can be made; highly explosive –exposure to direct sunlight should be avoided. Note : Nail, dense fibrous tissue, keratin masses, EMC need treatment with 4% phenol in 70% alcohol.

24. General Embedding Procedure 1. Open the tissue cassette, check against worksheet entry to ensure the correct number of tissue pieces are present. 2. Select the mould, there should be sufficient room for the tissue with allowance for at least a 2 mm surrounding margin of wax. 3. Fill the mould with paraffin wax. 4. Using warm forceps select the tissue, taking care that it does not cool in the air; at the same time. 5. Chill the mould on the cold plate, orienting the tissue and firming it into the wax with warmed forceps. This ensures that the correct orientation is maintained and the tissue surface to be sectioned is kept flat.

25. 6. Insert the identifying label or place the labeled embedding ring or cassette base onto the mould. 7. Cool the block on the cold plate, or carefully submerge it under water when a thin skin has formed over the wax surface. 8. Remove the block from the mould. 9. Cross check block, label and worksheet.

26. ORIENTATION OF TISSUE IN THE BLOCK Correct orientation of tissue in a mould is the most important step in embedding. Incorrect placement of tissues may result in diagnostically important tissue elements being missed or damaged during microtomy . Elongated tissues are placed diagonally across the block. Tubular and walled specimens such as vas deferens, cysts and gastrointestinal tissues are embedded so as to provide transverse sections showing all tissue layers. Tissues with an epithelial surface such as skin, are embedded to provide sections in a plane at right angles to the surface (hairy or keratinised epithelia are oriented to face the knife diagonally). Multiple tissue pieces are aligned across the long axis of the mould, and not placed at random.

27. SECTIONING/CUTTING It is the procedure in which the blocks which have been prepared are cut or sectioned and thin strips of varying thickness are prepared. The instrument by which this is done is called as a Microtome. Most microtomes use a steel blade and are used to prepare sections of animal or plant tissues for histology. TYPES OF MICROTOMES: Sliding Rotary Rocking Freezing Base sledge

28. ROTARY MICROTOME It is the most commonly used. Also known as Minnot’s Rotary microtome. In this the Block holder moves up and down while the knife remains fixed. It is suitable for cutting of small tissues & serial sections can be taken on it.

29. Parts of a Microtome ( Rotary ): A. Block holder B. Knife clamp screws C. Knife clamps D. Block adjustment E. Thickness gauge F. Angle of tilt adjustment G. Operating handle.

30. STEPS IN SECTIONING: Rough cutting of the block at 20 micron to expose the tissue. Side cutting of the block to get trim section and serial section. Put block in a bowl with ice cubes. Keep in referigerator (2 to 8 degree celsius ) for 1-2 hours. Cut the sections at 4 microns. Transfer the section on a water bath to remove wrinkles. Take section on albuminized slide. Before Staining, put the slides on hot plate to loosen the wax and fixation of issue on a slide.

31. STAINING Staining of the section is done to bring out the particular details in the tissue under study . The most commonly used stain in routine practice is Haematoxylin & Eosin (H&E) stain. H & E is a charge-based, general purpose stain. Hematoxylin stains acidic molecules in shades of blue(nucleus) Eosin stains basic materials shades of red, pink and orange(cytoplasm)

32. HAEMATOXYLIN & EOSIN(AUTO SLIDE STAINER) PROCEDURE Bring section to water. Stain with Haematoxylin for 8-10 minutes. Wash with water for 2-3 minutes. Differentiate in 1% acid alcohol for 15-18 seconds. Wash with water for 1 minute. Bluing in scott’s tap water for 3-5 minutes. Wash with water for 2-3 minutes. Stain with eosin for 45 seconds-3 minutes. Wash with water for 1 miute . Dehydration with alcohol for 1 minute- 3 changes. Clearing in Xylene for 3-5 minutes. Mounting Labelling Submission

33. MOUNTING Adhesives used for fixing the sections on the slides : Albumin solution ( Mayor’s egg albumin) Starch paste Gelatin Mountants : DPX ( Dibutylphthalate Polystyrene Xylene ) Canada Balsam Colophonium resin Terpene resin

34. TISSUE PROCESSING(MANUAL) PERCENTAGE AGENT USED DURATION 10% FORMAL SALINE OVERNIGHT 70% ALCOHOL 2 HOURS 90% ALCOHOL 1.5 HOURS ABSOLUTE ALCOHOL-I 1.5 HOURS ABSOLUTE ALCOHOL-II 1 HOUR ABSOLUTE ALCOHOL-III 1 HOUR - CHLOROFORM OVERNIGHT - TOLUENE-I 1.5 HOURS - TOLUENE-II 2 HOURS - TOLUENE-III 1.5 HOURS - PARAFFIN WAX-I 2.5 HOURS - PARAFFINWAX-II 4 HOURS

35. AUTOMATED TISSUE PROCESSOR All the before mentioned procedures upto the impregnation step can be done automatically in a single, unmanned instrument , which is the Automated Tissue processor. Advantages : It provides constant agitation during every step which ensures better fixation & processing. It reduces the work load & in turns improves the overall output of the laboratory.

36. RESTORATION OF SPECIMENS Keep the tissue in 70% alcohol (70ml), glycerol (80ml), and dithionite (1 gram). Leave the tissue in the solution in a sealed container, overnight. Perform the processing starting from the dehydration step.

37. Bancroft’s Theory and Practice of Histological Techniques (8 th edition) www.slideshare.net Image courtesy: GMSH pathology Department

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