Lecture (5) processing of tissue in histopathology laboratory

Tissue processing

1. Processing of Tissue in Histopathology Laboratory By: Ms. Hafsa sh. Hussein Histopathology & cytopathology lecturer Faculty of Laboratory, EAU. Lecture 5:

2. Purpose and aims of tissue processing

3. Provide sufficient rigidity to the tissue. If the tissue had enough rigidity then the tissue can be cut into thin section. Then used for microscopic examination .

4. Principle of processing: To remove water within the tissue. To impregnate paraffin wax in the tissue. Paraffin wax provides support to the tissue.

5. Tissue processing steps: Dehydration Clearing Infiltration & impregnation

6. Water must be removed from the tissue. Why ?? Dehydration:

7. Water doesn't mix with wax. Wet fixed  tissues  (in aqueous solutions) cannot be directly infiltrated with paraffin.

8. To clear/ remove the dehydrating agent The clearing agents is miscible to: Dehydrating agent. Impregnating medium. Clearing:

9. The tissue is infiltrated with a supporting medium. This supporting medium will provide adequate rigidity of the tissue . To make thin section for next step. Infiltration & impregnation:

10. Dehydration “ Every tissue contains some amount of free or unbound water molecule “

11. Things you should do in dehydration process

12. Dehydration should be done gradually from low to high concentration of dehydration fluid.

13. The tissue should be kept in the dehydration fluid for optimal time . Too much time  cause the tissue hard and brittle. Too little time  insufficient for removal of free water molecule

14. Smaller size need less time. Thin 2–3 mm tissue needs less time in dehydration fluid than Thick 5 mm tissue need more time.

15. Dehydrating agent

16. Ethanol Characteristics: The most commonly used. Clear and colorless fluid. flammable liquid. It needs license from the government to purchase ethyl alcohol for laboratory use.

17. Concentration: Use graduate ethanol (50, 70, 90 and 100%) For delicate/ soft tissue  started from 30% concentration. Use anhydrous cupric sulphate in final container Time: 2 h each Don’t immerse the tissue in the ethanol for long time. Causes hard and brittle tissue. Laboratory use:

18. Anhydrous cupric sulphate (CuSO4) White powder that draws water from the alcohol. Helps in dehydration.

19. How to use anhydrous cupric sulphate ? About 1 cm layer of this powder is kept in the bottom of the container. Then covered with 2-3 layers of filter paper. to prevent any colouring of the tissue. When the CuSO4 becomes hydrated, the colour of the powder changes to blue. This gives warning signal to change the alcohol and the CuSO4 powder.

20. Filter paper

21. Advantages: • Increases the life span of alcohol • Better dehydration • Good indicator to change alcohol

22. Methylated spirit known as denatured alcohol . Components: 99% ethanol 1% methanol / isopropyl alcohol) . Uses: Used in the same manner as ethanol.

23. Methanol Characteristics: Clear, colourless Volatile. Inflammable liquid. Rarely used in laboratory. its volatility and high cost.

24. Butyl alcohol n -Butanol isobutanol tertiary butanol Uses: Dehydrating agents in: Animal tissue processing. Plant histology processing.

25. Advantage: Tissue shrinkage is less than other alcohols.

26. Disadvantage: Slowly acting dehydrating agent. Takes longer time than ethanol.

27. Isopropyl alcohol Isopropyl alcohol is available as isopropanol (99.8%). This is miscible with water and liquid paraffin.

28. Advantages: Rapid acting dehydrating agent Non-toxic. Less tissue shrinkage. Good solvent.

29. Other Dehydrating agents

30. Dioxane 1,4-diethylene dioxide. Characterestics: Miscible with both water and molten paraffin wax .

31. Rapid dehydrating. Causes less tissue shrinkage. Tissue can be kept in dioxane without any harm. Advantages:

32. Liberates highly toxic gas Solution: Use proper ventilation. Disadvantages:

33. Ethylene glycol Other names: Ethylene glycol monoethyl ether Cellosolve . Characteristics: It is a colourless and odourless fluid.

34. Advantages: Rapidly acting dehydrating Tissue can be kept in Ethylene glycol for long duration.

35. Acetone Characteristics: Colorless Inflammable liquid Sharp ketonic smell. Miscible with: Water Alcohol

36. Advantages: Best used for fatty tissue processing.

37. Disadvantages: Causes tissue shrinkage Prolonged use may cause brittleness of tissue.

38. Clearing

39. Aims of clearing: Removal of dehydrating agent (e.g. alcohol) to facilitate impregnation of paraffin wax To make the tissue clear and improve the microscopic examination

40. Clearing is the process which leaves the tissues clear and transparent. Transparent tissue = complete dehydration. Any opacity of tissue = Incomplete dehydration. The amount of clearing agent should be 40 times of the volume of tissue for clearing.

41. Clearing agents

42. Xylene Characteristics: Most commonly used clearing agent in the laboratory. Clear Inflammable liquid.

43. Laboratory use: The small pieces of tissue are cleared rapidly by xylene Time: 30–60 min Note: Prolonged exposure to xylene may make the tissue hard and brittle.

44. Toluene Characteristics: Almost similar properties as that of xylene. Flammable. Action is slightly slower than xylene. Toxic. Prolonged exposure does not make the tissue hard.

45. Chloroform Characteristics: Highly volatile Non-inflammable Expensive. Toxic. Rarely used in laboratory.

46. Uses: Used in dense tissue. Example: uterus, muscle. Penetrating power: Chloroform is slower than xylene. Advantage: Does not cause any tissue shrinkage.

47. Esters Different esters: Amyl nitrate Methyl salicylate Methyl benzoate

48. Less toxic. Used in manual processing . Do not cause tissue hardening even under prolonged exposure.

49. Cedarwood oil Expensive Rapid clearing agent Used in clearing dense tissue .

50. Limonene Clear liquid. Advantage: Does not cause any tissue hardening. Disadvantage: Its removal from the tissue by paraffin wax is difficult .

51. How to select an appropriate clearing agent?

52. Type of tissue: large tissue takes more time. Type of processor: manual versus automatic. Processing condition: Temperature and vacuum

53. Speed of removal of dehydrating agent Ease of replacement by molten wax Safety factors: flammability and toxicity Cost

54. Books: " Basic and Advanced Laboratory Techniques in Histopathology and Cytology " by  Pranab Dey