Tissue processing in histopathology -4.pptx

Tissue processing in histopathology

1. TISSUE PROCESSING IN HISTOPATHOLOGY MODERATOR - Dr. Rashmi Sharma ma’am BY- Dr. Anand Kumar

2. TISSUE PROCESSING STEPS Tissue collection Fixation Dehydration Clearing 5. Impregnation 6. Embedding and blocking 7. Section cutting 8. Routine staining

3. PRINCIPLE In tissue processing the water within the tissue is removed , and another medium (usually paraffin wax) is impregnated in the tissue that provides the adequate support to the tissue. The basic aim of tissue processing is to provide sufficient rigidity to the tissue so that it can be cut into thin section for microscopic examination.

4. Influencing factors of tissue processing 1. Size of the tissue -The smaller the size the better the processing. -Optimum thickness of the tissue should be kept as 3 to 4 mm. 2. Heat -Increases the better penetration of fluid. -Excessive exposure to heat cause shrinkage and hardening of tissue. 3.Negative pressure -Negative pressure removes trapped air in the tissue.( eg -lungs ) 4.Agitation -This increases the flow of solutions around the tissue. 5.Viscosity -Dissolution with low viscosity has a faster penetration rate.

5. FIXA T ION Any tissue once taken out of the body will decompose due to:- Loss of blood supply and oxygen Accumulation of products of metabolism Action of autolytic enzymes Putrefaction by bacteria All the above changes PREVENTED BY FIXATION!

6. AIM OF FIXATION To preserve the tissue nearest to its living state. To prevent any change in shape and size of the tissue at the time of processing. To prevent any autolysis. To Make the tissue firm to hard.

7. To prevent any bacterial growth in the tissue. To make it possible to have clear stain. To have better optical quality of the cells.

8. COMMONLY USED FIXATIVES Formalin – MC – routine . Glutaraldehyde – electron microscopy . Picric acid (Bouin’s solution) – colour the tissue bright yellow in renal & testicular tissue . Alcohol(Carnoy’s fixative) – cytologic smears, endometrial sampling . Osmium tetraoxide – CNS tissues & electron microscopy . B - 5 – Lymph node biopsy

9. DEHYDRATION Dehydration displaces the residual fixative as well as cellular water. Water is present in tissue in 2 forms-, free and bound forms Free water removed by- graded alcohols. Bound water is removed by - heat or excessive time in the higher grade alcohols (95% or 100%).

10. 1. Ethanol -Ethanol or ethyl alcohol is the most popular and most commonly use dehydrating agent. 2. Methylated spirit (99% ethanol and 1% methanol) -It is also known as denatured alcohol. DEHYDRATING AGENTS

11. 3. Methanol -Highly inflammable liquid, volatile and high-cost so rarely use. 4. Isopropyl alcohol -It is a relatively rapid acting, non-toxic dehydrating agent causing minimal tissues shrinkage. -It is a good lipid dissolving solvent.

12. Incomplete dehydration will impair the penetration of the clearing reagents into the tissue leaving the tissue soft and non receptive to paraffin wax infiltration. Glycol ether dehydrates are an effective alternative to alcohol in tissue processing. Glycol ethers are unable to act as a secondary fixative like alcohols but their chemical properties prevent removal of bound water.

13. Colourless and inflammable liquid with a ketonic smell. Rapid action. Cheaper than ethanol. Good for fatty tissue processing. Prolonged use may cause shrinkage and brittleness of tissue. Image showing blocks being put in acetone for the DEHYDRATION . ACETONE

14. AIM OF CLEARING -Removal of dehydrating agent (e.g. alcohol) for impregnation of paraffin wax. -To make the tissue clear & improve the microscopic examination. IDEAL CLEARING AGENT -Low viscosity and high penetration rate. -Low melting point. -Miscible with both alcohol and molten wax. -No tissue damage. -Less toxic. -Less inflammable. -Cheap. CLEARING

15. Today, xylene is the most widely used clearing agent in tissue processing. It is an excellent lipid solvent but has the negative characteristic of drying tissue specimens. Total duration = Smaller biopsy : 1 hour. = Larger tissue: Three changes in xylene 60 minute each. Others Toluene, Chloroform, Benzene → Carcinogenic and toxic Cedar wood oil best but costly. XYLENE

16. Despite precautions taken during processing, technical or mechanical malfunctions and human error may occur, resulting in tissue drying out prior to paraffin wax impregnation. Tissue restoration can be accomplished by placing the tissue in a formol -glycerol solution for 5-10 hours. Whilst the tissue may not be ideal, this may provide slides of adequate diagnostic quality. RESTORATION OF DRIED TISSUE IN PROCESSING

17. IMPREGNATION/ INFILTRATION After clearing, tissue sections are infiltrated with paraffin wax to support the tissue, allowing thin sections to be cut. Infiltration must be sufficient to displace the clearant from the tissues otherwise wax will not harden properly. Clearing agent is removed by the process of diffusion .

18. IDEAL IMPREGNATING MEDIUM SHOULD HAVE FOLLOWING QUALITIES Miscible with clearing agent. Liquid in higher temperature and solid in room temperature. Homogeneous and stable. Non-toxic and cheap. Transparent. Fit for sectioning the tissue.

19. It is a type of hydrocarbon and it produced as a by-product during refining of crude petroleum. This is the most popular universally accepted embedding medium. In the Indian subcontinent the paraffin wax with melting point around 60°C is the most suitable for laboratory use. Total 3 to 4 hours time in paraffin wax is sufficient for impregnation of tissue. ADVANTAGE OF PARAFFIN WAX Tissue block can be stored for long duration. Non-toxic. Cheap and Safe. DISADVANTAGES OF PARAFFIN WAX It may cause tissue shrinkage and hardening in case of prolonged impregnation. It takes long duration for the impregnation of the bone and Eye. PARAFFIN WAX

20. Resin Used exclusively as the embedding medium for electron microscopy, ultrathin sectioning for high resolution and undecalcified bone. Agar It does not provide sufficient support for sectioning tissues. Its main use is as a cohesive agent for small friable pieces of tissue after fixation, a process known as double embedding , when fragments of tissue are embedded in melted agar, allowed to solidify and trimmed for routine processing. Gelatin This is primarily used in the production of sections of whole organs. Celloidin It is used when processing dense and/or hard tissues. ALTERNATIVE INFILTRATION MEDIA

21. TISSUE PROCESSOR Dehydration + C learing + I mpregnation Automated tissue processor The basic principle of tissue processor is to transfer the tissue in different fluid for a specified time in a desired environment. 12 stations 1 jar – formalin 6 jars – grades of alcohol 3 jars – xylene 2 jars – molten paraffin wax

22. In this system the bucket of tissue is transferred from one container to other after a specified time. Tissue remains in a basket with 30 to 100 cassettes. The basket containing the tissue is submerged in the specific container for a particular time and then transferred to the next container automatically. A gentle agitation is created by vertical oscillation or by rotatory movement of the tissue basket. The time schedule and transfer of tissue in each container are determined by microprocessor.

23. In our histopathology lab manual tissue processing is done and graded acetone is used. TIME SCHEDULE FOR TISSUE PROCESSING MANUAL TISSUE PROCESSING

24. Small number of samples can be processed. Careful monitoring in each step is possible. In case of emergency when the automated tissue processor is not working, one can take the help of the manual processing. In case of manual processing, it is possible to select the reagents of choice with flexibility in time duration. ADVANTAGE OF MANUAL PROCESSING ARE

25. MICROWAVE PROCESSING Microwave processing in histopathology reduce the time of processing significantly. It is suitable for small number of delicate tissues. Microwave oven generates heat within the tissue which warms the tissue block in short period of time. The microwave oven usually has : System to control the temperature. System to control the time duration of particular temperature. Proper exhaust to remove the toxic gas.

26. EMBEDDING & BLOCKING Process of creating tissue blocks by using an external support medium to enable microtomy . AIM OF EMBEDDING To give support to the tissue. To prevent distortion of the tissue during cutting. To preserve the tissue for archival use. Embedding – with molten wax.

27. EMBEDD I NG CENTRE Wax reservoir . Heated area for steel moulds . Wax dispenser . Separate hot and cold plates .

28. PLASTIC EMBEDDING RINGS DISPOSABLE PEEL AWAY MOULD STAINLESS STEEL MOULD TISSUE CASETTE

29. PARAFFIN WAX EMBEDDING STEPS

30. Moulds are put on cold plate Remove casette from mould Now blocks are ready for sectioning ( Microtomy )

31. TISSUE ORIENTATION The correct orientation of the tissue is very important for proper cutting and microscopic examination. Tissue is usually placed as flat on the central part of the mould. It should be oriented in such a way so that cutting is easy by knife of the microtome. The tubular tissue- like fallopian tube, vas deferens , artery etc should be placed in such a manner so that we get a transverse section with all the layers

32. Tissue with epithelial surface should be placed vertically and right angle to the surface so that we can get all the layers. Multiple section of the tissue such as endometrial curettage should be placed all in central position. Linear long tissue should be placed diagonally.

33. Muscle biopsy should be placed both in longitudinal and transverse plane. Long membranous tissue such as amniotic membrane should be made as swiss roll.

34. SE C TION C U TTING It is the procedure in which the blocks which have been prepared or cut or sectioned, from that thin strips of varying thickness are prepared. The instrument by which this is done is called as microtome. Microtome is used to make the thin slices of tissue usually 4um but can be 2 to 10 um. 5 types of microtomes : Rotary– M/C used Sliding Freezing Rocking Base sledge

35. 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 and serial sections can be taken on it.

36. Sliding microtome Knife or blade is stationary and the specimen slides under it during sectioning. This microtome was developed for use with celloidin embedded tissue blocks, used primarily for research. Rocking microtome Commonly used in cryostats, the retracting action moves the tissue blocks away from the knife on the upstroke, producing a flat face to the tissue block. Ultra microtome Used exclusively for electron microscopy.

37. After microtomy we put this section slice in the water bath. It is the thermostatically controlled water bath with inside coloured black. It is maintained at a temperature of 5 to 6° below the melting point of paraffin wax So wax will not melt. This step is done to remove creases and folds in sections slice. TISSUE FLOTATION (WATER) BATH

38. After the water bath we take these floating tissues on glass slide. The slide is coated with egg albumin so that tissue can adhere on that slide.

39. Methods used to produce sections which preserve cellular morphology without use of dehydrating and clearing solutions and heat. Frozen section Uses of frozen sections Intraoperative diagnosis. Diagnostic and research histochemistry for labile enzymes. Immunofluroscence . Immunohistochemistry techniques when heat and fixation may inactivate or destroy the antigens. Diagnostic and research non- enzyme histochemistry , e.g. lipids and some carbohydrates.

40. PRINCIPLE OF FROZEN SECTION When the tissue is frozen, the interstitial water in the tissue turns to ice and in this state the tissue is firm, the ice acting as the embedding medium. The consistency of the frozen blocks may be altered by varying the temperature of the tissue. Reducing the temperature will produce a harder block ; raising the temperature makes the tissue block softer.

41. PREPARATION OF FROZEN SECTION Fix small blocks of tissue in 10% buffered neutral formalin. Remove fixative by washing specimen in water before cutting. Place a drop of water on the specimen holder and place the block in position parallel to the knife edge. Apply gentle downward pressure on the tissue with a glass slide and release freon until specimen is frozen. Currently there are available embedding compounds which will assist in cutting, these are called O.C.T (Optimum cutting temperature) compounds which provide a surrounding matrix that holds the block and prevents shattering. If frozen section is extremely fragile or of small fragments of exudate are to be cut, the material can be embedded in gelatin. Start sectioning until a complete section is obtained.

42. 6. Lift the section from the knife and place the section in a dish of distilled water. Sections may be picked up on albuminized slides and dried before staining. 7. The tissue should be immediately fixed in methanol for 1 minute or 95% ethanol for few seconds. Rapid fixation within few seconds is mandatory. In case of delayed fixation, cells are swollen, and the cytoplasmic margin may be ruptured giving hazy appearance. 8. The slide is rinsed in tap water and then stained with haematoxylin and Eosin.

43. The Cryostat

44. Staining of the section is done to bring out the particular details in the tissue under study. ROUTINE STAINING (H&E) The most commonly used stain in routine practice is Haematoxylin & eosin stain. Haematoxylin (Basic dye) – Nuclear stain (Acidic) Eosin (Acidic dye) – Cytoplasmic stain

45. Deparaffinization with xylene. The section of tissue themselves have paraffin wax around, so slightly warm the slide and dip in xylene for 1-2 minutes, to remove wax and only tissue stick to slide, this step is called deparaffinization . Hydration In the starting of the tissue processing we remove the water from the tissue, but stains are water soluble so it is required for staining. In this we put tissue in higher to lower grade of alcohol and finally to distil water. PROCEDURE

46. Stain with Haematoxylin for 15 min Wash with running water Differentiate with 1 % acid alcohol Wash with running water for 5 mins Stain with 1% Eosin for 2 min Wash with water Dehydration with graded alcohol Clearing with xylene 3-5 min Dry & Mount with DPX

47. End result :-

48. SPECIAL STAINS special stains are used to identify certain normal and abnormal substance present in the cell and tissue ,which can not be identified on routine haematoxylene & eosin staining or better appreciated on special stain. PAS(PER IODIC ACID SCHIFF) Glycogen Fungal hyphae Basement Membrane Lymphoblast-block positivity AMYLOID Congo Red Methyl violet Crystal Violet Thioflavin T

49. IRON - Prussian Blue/Perl’s Stain CALCIUM von kossa Alizarin Red S Calcein TB - ZN Stain Masson Trichrome - Collagen fibres, muscles Von Gieson stain – Collagen Mucicarmine stain – Mucin of intestinal adenoca , capsule of cryptococci FAT oil red o Sudan black B MELANIN - Masson Fontana H.PYLORI - Warthin Starry Silver Stain