Tissue processing ppt with full explanation

Tissue processing

1. TISSUE PROCESSING Presenter: Dr. Pranjal Moderator: Dr. Pruthvi D.

2. OBJECTIVES Prerequisites Principle of Tissue Processing Stages of T issue Processing Types of Tissue Processing Embedding Tissue Microarray

3. INTRODUCTION Tissue processing is defined as the stepwise treatment of tissues to remove all extractable water from the tissue and impregnation of the tissue with a solid support medium that is firm enough to support it and give sufficient rigidity to enable thin sections and that is soft enough not to cause damage to the knife or tissue.

4. PRINCIPLE A B C Fixation Embedding Dehydration, clearing

5. FACTORS INFLUENCING RATE Specimen size Agitation Heat Viscosity Vacuum Ultrasonics .

6. PRE-REQUISITES Proper receipt and identification of the specimen should be done. Proper sections of tissue should be taken, not exceeding 3x2x0.4 cm. Proper labeling should be done of each section of the tissue before putting it into the cassette.

7. STAGES OF TISSUE PROCESSING Fixation Dehydration Clearing Impregnation Embedding

8. FIXATION Process that preserves the morphological and chemical characteristics of cells and tissues and prevents autolytic and putrefactive changes. It stabilizes and hardens the tissue with minimal distortion of cells.

9. PROPERTIES OF FIXATIVES Cheap and easily available Stable and safe to handle Should cause fixation quickly Minimal damage to the tissue Even penetration of the tissue Retain the colour of the tissue

10. FIXATIVES Simple fixatives Compound fixatives

11. SIMPLE FIXATIVES Formalin Glutaraldehyde Osmium Tetroxide Potassium Dichromate Mercuric Chloride

12. COMPOUND FIXATIVES Formalin-based fixatives Mercurial fixatives Dichromate fixatives Picric acid fixatives

13. FORMALIN 10% Most commonly used fixative. Contains 10 ml of 40% formaldehyde with 90 ml of water. Reacts with proteins and forms cross-links between the protein molecules.

14. ADVANTAGES Cheap, easy to prepare and stable. Tissue penetration is good. Best fixative for nervous tissue. Allows subsequent use of most staining procedures including IHC. Natural tissue colour restored after fixation.

15. DISADVANTAGES Dermatitis Damage to nasal mucosa Formation of dark-brown formalin pigment May be carcinogenic Gradual loss of staining on prolonged fixation.

16. GLUTARALDEHYDE Aldehyde fixative with slower rate of diffusion than formaldehyde but faster fixation. Mainly used for electron microscopy. Morphological features are well preserved. Best cross-linking agent for collagen. False PAS positivity.

17. MERCURIC CHLORIDE Advantages: Excellent staining of nuclei and connective tissues. Quick fixation. Staining of cytoplasm is more brilliant. Gives best results with metachromatic staining. Best preservation of details for photomicrography.

18. MERCURIC CHLORIDE Disadvantages: Not recommended as fixative for demonstration of nucleoproteins or sulfhydryl groups. Rate of penetration reduces after first few mm. Excessively hard and brittle tissue on prolonged fixation. Corrodes all metals except Monel . Brownish mercurous chloride precipitate.

19. POTASSIUM DICHROMATE Advantages: Used for fixation of mitochondria. No precipitation. Disadvantages : Direct transfer to alcohol causes formation of insoluble oxide. So, needs to be thoroughly washed in running water.

20. DEHYDRATION Water in aqueous fixatives as well as in tissues are immissible with paraffin wax and so has to be removed. Dehydration is the process of removal of water and fixative from the tissue. Achieved by immersing in increasing grades/strengths of alcohol.

21. DEHYDRATING AGENTS Ethyl alcohol Methyl alcohol Isopropyl alcohol Butyl alcohol Tetrahydrofuran Acetone Dioxane

22. ETHYL ALCOHOL Fast-acting, clear, colorless, non-poisonous, flammable and reliable liquid. Best dehydrating agent, produces total dehydration and replaces aqueous fixative and unbound water. Dehydration should be complete. Dehydration is carried out using ascending grades of concentrations.

23. Advantages Disadvantages Non-toxic Hardening of tissue if left for long periods Graded alcohols starting from 70% if used, avoids shrinkage of tissues. High excise duty, so expensive and needs license Ideal dehydrating agent for delicate tissues Requires 3-4 changes

24. METHYL ALCOHOL A clear, colorless and flammable liquid which is miscible with water, ethanol and most organic solvents. Highly toxic.

25. ISOPROPYL ALCOHOL Miscible with water, ethanol and most organic solvents. Mainly used in microwave processing schedules.

26. Advantages Disadvantages Easily available Expensive Does not produce overhardening or shrinkage of the tissue Cannot be used in celloidin techniques Fast acting, non-toxic and reliable.

27. ACETONE Clear, colorless and flammable liquid Miscible with water, ethanol and most organic solvents.

28. Advantages Disadvantages Good, rapid dehydrating agent Highly volatile and inflammable and shrinks the tissue. Cheap, readily available Tends to harden and produce brittleness on prolonged dehydration Easily removed by most clearing agents Poor penetration and removes lipids from tissue during processing

29. Advantages Disadvantages Mixes freely with water, paraffin, xylene and alcohol Expensive than alcohol and odorous Acts as both a fast dehydrating and a clearing agent Has a cumulative toxic action Less shrinkage and hardening of tissue Areas of use should be well ventilated DIOXANE

30. TETRAHYDROFURAN (THF) 50% THF for 2 hours 100% THF for 3 changes of 2 hours each Equal parts of THF and wax for 2 hours Another 2 hours in wax.

31. CLEARING/DEALCOHOLIZATION Acts as an intermediary between the dehydration and infiltration solutions. Should be miscible in both the dehydrating agent as well as in the infiltrating solutions. When completely replaced by clearing agent, tissues appear transparent, so this process is called clearing.

32. PROPERTIES OF A GOOD CLEARING AGENT Rapid or quick removal of dehydrating agent. Rapid penetration of tissues. Clear the tissue quickly without hardening or tissue damage. Easily removed by molten paraffin wax. Low flammability, toxicity and cost. Does not evaporate too quickly in the wax baths.

33. CLEARING AGENTS Xylene Toluene Chloroform Benzene Carbon tetrachloride Cedar-wood oil

34. CLEARING AGENTS Citrus fruit oils Paraffin wax Histoclear CNP 30 and inhibisol Propylene oxide

35. XYLENE Flammable, colorless liquid with characteristic petroleum odor. Miscible with most organic solvents and paraffin wax. Most commonly used clearing agent in routine histopathology. Refractive index is 1.50.

36. ADVANTAGES Rapid clearing agent Fairly cheap and easily available Makes the tissue transparent, end point of clearing can be easily identified.

37. DISADVANTAGES Volatile and highly inflammable, vapor is an irritant. Prolonged treatment hardens the tissue and makes it brittle. If xylene is added before completion of dehydration, it becomes milky. Not advisable for brain and lymph nodes (too brittle).

38. TOLUENE More volatile and flammable than xylene Advantages : Does not harden tissue. Disadvantages : Inflammable and potentially dangerous. Clears tissue less rapidly than xylene.

39. BENZENE Advantages : Rapid action, little shrinkage, no hardening of tissues. Evaporates quickly. Disadvantages : Highly flammable. Causes aplastic anemia and cancer in the laboratory worker.

40. CHLOROFORM Advantages : Tissue can be left longer without rendering them brittle. Minimal shrinkage and hardening compared to xylene. Ideal for brain and lymph nodes. Nonflammable.

41. CHLOROFORM Disa dvantages : Highly toxic (phosgene). Highly expensive. Slow in its action (6-24 hrs ). No change in refractive index, end point of clearing cannot be made out. Anesthetic.

42. CITRUS FRUIT OILS Limonene reagents Derived from orange and lemon peels. Non-toxic and miscible with water. Can cause sensitization. Strong pungent odor. Oily and cannot be recycled.

43. CEDAR-WOOD OIL Advantages No tissue shrinkage. Does not dissolve out aniline dyes. Good penetration. No damage or hardening of tissues. Best clearing agent.

44. CEDAR-WOOD OIL Disa dvantages Wax impregnation is slow. Highly expensive. Toxic and more viscous.

45. TECHNIQUE OF CLEARING BY XYLENE Remove tissue from last beaker of alcohol. Place in 2 changes of xylene for 30 mins each. Transfer to paraffin wax.

46. INFILTRATION Permeation of the tissue with a support medium. Process of replacing the clearing agent by an embedding medium. Helps in cutting thin sections easily.

47. EMBEDDING REAGENTS Paraffin wax Paraplast Paraplast plus Water soluble wax Polyester wax Micro-crystalline wax

48. EMBEDDING REAGENTS Resins Celloidin Agar Gelatin Carbowax

49. PARAFFIN WAX Most commonly used, cheap. White or colorless soft solid composed of long straight-chained hydrocarbons. Permeates the tissue in liquid form, solidifies rapidly at room temperature. No distortion of tissue, handled and stored with ease.

50. MELTING POINT OF PARAFFIN Defined as the temperature at which a drop of molten wax becomes semisolid on the bulb of a slowly rotating thermometer. Ranges from 47 to 64 degree celsius . Usually 58 to 60 degree celsius is preferred.

51. ADVANTAGES OF PARAFFIN Inexpensible . Provides sections of good quality. Easily adaptable to a variety of uses. Compatible with most routine and special stains.

52. PARAFFIN ADDITIVES Ceresin Bees wax Micro-crystalline wax Bayberry wax

53. PARAFFIN ADDITIVES- PURPOSE To cut thinner sections Increase hardness To get good ribbon sections. To alter the crystalline structure of wax to improve sectioning.

54. OVENS/INCUBATORS Impregnation with paraffin wax takes place in an incubator. Should have a temperature range of 50-60 degree celcius . Should be large enough to accommodate an enamel jar, one or two Coplin jars and few containers for wax impregnation of tissues.

55. STEPS OF IMPREGNATION After blotting lightly with filter paper, tissue is transferred into molten paraffin wax. Tissue should pass through at least 2 changes of wax. Impregnation with embedding media takes place in a thermostatically controlled oven. Temperature maintained at 2-3 degree celcius above the melting point. Volume of wax: 25-30 times the volume of tissue.

56. DURATION OF IMPREGNATION Depends on:- Size of tissue: Thicker tissue requires more time. Type of tissue: Dense tissue (bone, skin, CNS) require more time. Clearing agent used: Xylene, toluene, benzene require less time; Cedar-wood oil require more time and several changes. Vacuum embedding oven: Reduces time required.

57. TYPES OF PARAFFIN WAX Liquid paraffin: used as oil for hematology, mounting media for frozen section. Liquid paraffin with R.I. of 1.48: used for spectroscopy. Petroleum jelly: Lubricant. Soft and white-melting point 56-60 degree celcius : Histopathology.

58. ALTERNATES Paraplast Paraplast plus Water soluble waxes Resin Agar Celloidin Gelatin Plastic embedding media

59. PARAPLAST More elastic than normal paraffin wax (superior to double embedding). Does not need cooling before cutting. Same melting point as routine paraffin wax. Represents a major technical advancement for routine and research work.

60. PARAPLAST PLUS Contains DMSO (dimethyl sulfoxide ). Allows more rapid penetration. Reduces the time for tissue processing. No need for filtering.

61. WATER SOLUBLE WAXES Polyethylene glycols with melting point of 38-40 degree celcius . Can be directly embedded from water. Used for demonstration of lipids and enzymes.

62. RESIN Embedding medium for electron microscopy Ultra-thin sectioning can be done. Undecalcified bone can be embedded.

63. AGAR Used as a cohesive agent for small friable pieces of tissue after fixation, a process called as double embedding.

64. CELLOIDIN Purified nitrocellulose. High resilience, hard or fragile tissues are more easily cut. Heat is not needed during processing- minimal shrinkage and distortion. Impregnation takes 2-3 weeks.

65. GELATIN Water-soluble wax. Used in sections of whole organs.

66. DOUBLE EMBEDDING Double embedding is a technique in which the tissue is first impregnated with celloidin and subsequently blocked in paraffin wax. Serial sections can be easily prepared. Extra degree of resilience is added. A tedious method.

67. TYPES OF TISSUE PROCESSING Hand processing Automated processing

68. AUTOMATED TISSUE PROCESSING Place the solution and paraffin in respective beakers. Timing leaver is set at 0. Baskets with the cassettes automatically change position and takes a bath in different reagents kept in different beakers in respective order. Casettes are opened next morning for embedding.

69. OVERNIGHT PROCESSING SCHEDULE Sl. No. Reagents Duration 1. 10% Formalin I 1 hour 2. 10% Formalin II 1 hour 3. 50% alcohol/formalin 1 hour 4. 70% alcohol 1 hour 5. 95% alcohol I 1 hour 6. 95% alcohol II 1 hour 7. Absolute alcohol I 1 hour 8. Absolute alcohol II 1 hour 9. Xylene I 1 hour 10. Xylene II 1 hour 11. Paraffin I 2 hours 12. Paraffin II 2 hours

70. PREPARING PARAFFIN BLOCKS Leuckhart’s “L” molds Plastic embedding rings Plastic ice trays Paper boats Embedding trays Glass petridishes

71. LEUCKHART’S “L” MOLDS L-shaped brass pieces placed in opposing positions and can be manipulated to modify the size of block to be prepared.

72. SECTION CUTTING Procedure of cutting and sectioning prepared blocks to obtain thin strips of tissue. Instrument used is called a microtome.

73. TYPES OF MICROTOME Sliding Rotary Rocking Freezing Base sledge

74. TISSUE MICROARRAY Method used to evaluate numerous samples of tissue in a short period of time. Multiple tissue samples can be arranged in a single paraffin block using precision tools to prepare the recipient block.

75. TECHNIQUE A hollow needle is used to take 100 or more tissue core samples from specific areas of pre-existing blocked tissue. Placed in a single array block. Sections are then taken from this block. Thus, a single slide containing hundreds of tissue cores can be obtained.

76. USES IHC In-situ hybridization FISH Special stain control samples. Quality control sections for H&E.

77. ADVANTAGE Cost-effective: only a small amount of reagent used per slide.