FROZEN TECHNIQUES .pptx

post graduate ppt on cryostat

1. PRESENTER: DR. N. MANJULA POST GRADUATE DEPT OF PATHOLOGY FROZEN TECHNIQUES

2. CONTENTS INTRODUCTION. PRINCIPLE. INDICATION. CRYOSTAT MACHINE. STAINING. ADVANTAGE. DISADVANTAGE. ARTIFACTS. TROUBLE SHOOTING.

3. INTRODUCTION The frozen section procedure is pathological laboratory procedure to perform rapid microscopic analysis of a specimen. Frozen section resulted from need for rapid intra operative diagnosis. Louis B. Wilson pioneered the frozen section at the Rochester in 1905. It is used most often in oncological surgery.  The technical name for this procedure is  CRYOSECTION .

4. Louis B. Wilson pioneered the frozen section.

5. PRINCIPLE The basic principle in frozen section is to freeze the tissue so that the water within the tissue transforms into ice. The tissue becomes firms. This ice acts as the embedding medium . We can adjust the consistency of the frozen block by adjusting the temperature of the tissue. Higher temperature is required to make the fixed block of tissue to soften and get good sections .

6. INDICATIONS OF FROZEN SECTIONS

7. INSTRUMENTS USED IN FROZEN SECTION FREEZING MICROTOME : It consists of fixed stage over which the knife moves. Here CO2 is used to freeze the tissue. The microtome stage is hollow and perforated at sides to allow free flow of CO2. CO2 absorbs heat and rapidly freezes the tissue. Wedge shaped knifes are used.

8. FREEZING MICROTOME

9. THE CRYOSTAT The cryostat was introduced in 1954 and there have been improvements in the instrument which facilitated both safety and section cutting. The cryostat is the instrument that has the arrangement to freeze the tissue and also to cut the frozen tissue for microscopic section. The cryostat is a refrigerated cabinet and microtome is placed inside. Presently, rotary microtome is the type of choice.

10. LABELLING THE MACHINE

11. This microtome is controlled from outside the cabinet. Modern cryostats are caster mounted cabinet models of the open-top type which permits easy access and visibility through the double thickness, hinged plastic cover. ROTATORY MICROTOME REFRIGERATED COMPARTMENT

12. REFRIGERATED COMPARTMENT OPEN-TOP CABINET

13. The rust proof microtome is usually mounted at 45° angles in the stainless steel cabinet. This cabinet is equipped with an antifogging air circulating system, a drain for defrosting and sterilization and a shelf with spaces for 4–6 metal block carriers.

14. Angle 45 Shelf

15. FACILITIES OF CRYOSTAT Electronic control of temperature. Electronic control of advancement and retraction of tissue block. Digital visualization of cabinet temperature. Mechanical control for cutting speed and section thickness. Automatic defrost mechanism. Automated decontamination and sterlization .

16. TYPES OF CRYOSTAT Closed cycle cryostat. Continuous flow cryostat. Bath cryostat. Multistage cryostat.

17. THE COMMON FREEZING TECHNIQUES Liquefied nitrogen (–190°C) is commonly used. It is the most rapid freezing agent. ™Isopentane cooled by liquid nitrogen (–150°C) ™Carbon dioxide gas (–70°C) ™Carbon dioxide (–70°C) ™Aerosol sprays (–50°C)

18. Temperature range in the machine : The cryostat machine has the usual temperature range from 0 °C to −35 °C. The most of the tissue is sectioned properly between −15 °C and −25 °C. The water-containing tissues can be sectioned in higher temperature, and fat-containing tissue needs much lower temperature to cut. Rotary microtome : Rotary microtome is placed inside the cabinet of the cryostat. Here the knife is fixed and the tissue is moved with the help of a rotary wheel.

19. Tissue shelf : Just in one side of the microtome, there is a tissue shelf to keep the tissue. In this place the samples are kept for freezing. Usually the temperature of tissue shelf is lower than the overall cabinet temperature. Specimen holder : The specimen holder or chuck is supplied by the manufacturers in different sizes and shapes. Usually these are round metal structures.

20. TISSUE IN HOLDER Tissue shelf

21. While embedding tissue for frozen the surface which we need to study faces up while for normal paraffin embedding blocks the surface faces down.

22. Knife or blade : Nowadays, low- or high-profile disposable blades are used. The blade should be proper fixed to the holder to get an even pressure in the whole length. The angle of the knife is kept in between 5° and 7°.

23. Antiroll plate : Just in front of the knife, there is an antiroll plate that prevents the rolling of the cut tissue. It is the natural tendency of all frozen section to curl upwards on sectioning in the cryostat. Anti roll plate prevents it and guides the frozen section towards down. It is usually a glass plate within a metal frame. The undersurface of the plate has free space, and there is a gap between the knife and the plate. Alternating to antiroll plate, a cool sable hair brush can be used to get unrolled tissue.

24. BLADE microtome Antiroll plate

25. PLACE TO KEEP THE BRUSH AND KNIFE HOLDER Place to keep the brush and knife holder : Just in front of the microtome machine, there remains a small place to keep the brush and knife holder.

26. Embedding medium : This medium is used to hold the tissue over the chuck. Presently optimum cutting temperature (OCT) compound is used as embedding medium. The OCT is made of water-soluble glycols and resin.

27. WHAT PATHOLOGISTS SHOULD KNOW DURING FROZEN Whether tissue selection is appropriate or not. Type of tissue cut at which temperature. Time consideration ( 20 – 30 min ), ideally from sample receiving to reporting. Communication with surgeon.

28. Cryostat Sectioning The process of the cryostat sectioning needs the following steps. Grossing and cutting the specimen : The cutting surface of the tissue should be smooth. The following steps in grossing of the tissue are mandatory for accurate reporting: Identify the tissue sample of the patient and the requisition form : This is the first and foremost part of the frozen tissue grossing.

29. REQUEST FORM FOR FROZEN

30. GROSSING FOR FROZEN SECTION TISSUE

31. Salient clinical information : The essential clinical information is very helpful as it guides the pathologist to reach the possible differential diagnosis. Tissue appearance : The gross appearance of the tissue such as colour, texture, consistency and any suture to mark the anatomical position. Resection margin : It is very important to identify the resection margins of the tumour. The resection planes and margins should be inked thoroughly. Different colours of ink can be used for medial and lateral margin identification.

32. Cutting the tissue : The tissue should be fresh without any fixative. The tissue should be preferably dry, and it should not be wrapped in gauze piece. Any suture, staple or sharp hard structure should be removed from the tissue sample. Now the tissue is cut into small pieces as it facilitates freezing. Take multiple sections of the tissue to understand the main pathology and to minimize the error. Use a new sharp scalpel blade, and first cut the most important area that needs microscopic examination. It is preferable to use gentle stroke of the scalpel rather than too much pressure.

33. Cytology of the tissue : At times the imprint of the tissue on the slide provides good morphological details such as lymphoma of the lymph node. Similarly crushing of tissue also provides excellent morphological details such as in case of tissue of the brain tumour. Tissue embedding in the mould : The small piece of the tissue is kept in the centre of the mould, and then the OCT is poured over it in excess. Then the tissue holder or chuck is firmly placed over the tissue with overflown OCT.

34. CYTOLOGY PREPARATION: IF NEEDED MAKE

35. Tissue loading in the frozen section chamber : The tissue is now placed in the frozen section chamber, and cold spray can be used to make the process faster. Loading the blade : The cutting knife or blade is now loaded and the proper alignment is done. Trimming the tissue : The loss of normal or natural colour to whitish colour indicates that the tissue is frozen. The frozen tissue in the tissue holder is now placed in the holder of the microtome. The block is trimmed to remove the excess OCT and to get the smooth tissue surface for sectioning.

36. Sectioning : The tissue is now cut gently and is spread over the antiroll plate with the help of a brush. The brush should be cooled. The tip of the tissue is guided by the brush. Section lifting : The glass slide of normal room temperature is pressed firmly over the tissue section, and normally the tissue sticks immediately.

37. REGISTER MAINTAINED FOR FROZEN

38. Factors Affecting the Good-Quality Section The common factors responsible for the good-quality smear include: Temperature : When the temperature falls, water within the tissue becomes frozen and gives the tissue hard consistency. The optimal temperature of frozen tissue is in between −15 °C and −25 °C. Warm tissue remains soft and sections crumple. On the other hand, the overcooled tissue becomes very hard and brittle and produces again bad-quality crumpled section.

39. OPTIMUM TEMPERATURE FOR FROZEN SECTION

40. Moreover the hard tissue may cause “chattering” artefact and also thick and thin sections. Different tissue contains variable amount of fat and water. The consistency of different tissue varies, and therefore the optimum temperature to cool the tissue varies considerably.

41. Tissue consistency : The consistency of tissue has significant effect on cutting such as: Fatty tissue : It is difficult to cut the fatty tissue in frozen section. Fat may smear on the knife and may make problem in cutting. Collagenous tissue : The firm collagenous tissue is difficult to cut. Necrotic tissue : Soft necrotic tissue may create considerable problem as they may fall from the slide making hole in the section. It is preferable to take only viable tissue for frozen section.

42. Bony hard tissue : Hard tissue like bone or cartilage may damage the blade significantly. In this situation a new section can be processed, or new blade can be used. Tissue size : The size of the tissue sample should be small as the larger tissue takes much longer time to freeze.

43. FIXING CRYOSECTION TO SLIDES Fresh, unfixed tissue usually does not require adhesive on slides. But formation or otherwise fixed tissue may not adhere to slides and therefore may detach during staining. Albumen or Zwemer’s chrome-glycerol jelly may be used in these cases. Poly-L-lysine and gelatin -formaldehyde mixture may also be used. The 40°C difference between cryostat section (–18 to –20°C) and slide (room temperature 37°C) is usually sufficient to attach the section to slide as well as producing some heat fixation.

44. ALBUMIN MICRON TEMPERATURE TIME SLIDES

45. STAINING OF CRYOSECTION rapid h and e

46. HEMATOXYLIN EOSIN WATER BATH

47. ADVANTAGES

48. DISADVANTAGES

49. FROZEN TISSUE PARAFFIN EMBEDDED TISSUE 1. FIXATION Pre/Post-sectioning Pre-embedding 2. SECTIONING Cryostat Microtome 3. STORAGE Usually up to one year at –80°C. Several years at room temperature. 4. ADVANTAGE Preserves certain enzymes and antigen functions Preserves tissue morphology 5. LIMITATIONS Formation of ice crystals may adversely affect tissue structure Over fixation can mask the epitopes 6. PRECAUTIONS Tissue should not be frozen slowly to prevent ice-crystal formation. Tissues should be allowed to warm to cutting temperature (–20 to –22°C) in cryostat to avoid shattering. Duration and intensity of tissue heating should be kept to a minimum as melting temperature of paraffin wax (50–60°C) may be deleterious to staining of some antigens. 7. ROUTINE USE • Rapid diagnosis and status of margins (involved or not) during operation • Fat stain, enzyme demonstration and some other special stains need cryostat sections. • Histologic sections, subsequent staining (H and E and others) and microscopical examination for confirmatory diagnosis of tumors , infections and other pathologic lesions • Also useful to demonstrate normal histology and to make control sections.

50. FROZEN H& E 100 X PERMANENT SECTION H & E 100 X

51. FROZEN H& E 400 X PERMANENT SECTION H & E 400 X

52. ARTIFACTS ICE CRYSTAL ARTIFACTS: Slow freezing of tissue. OVER FREEZING Cause sections to have holes. Tissue with high water content Eg : edematous / bloody tissue.

53. ULTRACRYOTOMY It is mainly used in research labs. Tissues are freezed using isopentane / liquid nitrogen. Sections of 50 – 150 nm are cut. For better results tissues are fixed in glutaraldehyde prior to sectioning. Temperature maintained is between -20 to -212 C. Sections are cut using glass knife. These sections are used for localization of enzyme activity at the ultrastructural level.

54. FREEZE DRYING It is the technique of rapid freezing of fresh tissues at -160 C with subsequent removal of water molecules in form of ice by sublimation in a vacuum at higher temperature of – 40 C. Blocks are then raised to room temperature by vapor . This technique is restricted to research labs. It minimizes loss of soluble substances, displacement of cell contents, denaturation of proteins, inactivation of enzymes.

55. FREEZE SUBSTITUTION This technique involves rapid freezing of tissue to – 160 C in isopentane super cooled by liquid nitrogen. Sections are cut at 8 – 10 micron and placed in a cold container maintained at cryostat temperature. The sections are transferred to water free acetone and cooled to – 70 C for 12 hours. The sections are floated onto slides, allowed to dry and required histochemical method is applied.

56. QUALITY ASSURANCE Never two cases in one grossing area. System of identification of block and slide should be established. Never two cases in one cryostat. Unlabelled slides should never be used. Cryostat has to be cleaned periodically. Records must be maintained properly.

57. MISUSE OF FROZEN Sometime the procedure is misused by surgeon to satisfy their curiosity. To compensate their deficiency in recognizing normal anatomical structure. To communicate the results to patient’s relative.

58. POINT TO REMEMBER Definitive diagnosis is not possible in all cases . Important for surgeons to influence the mode of surgery. Misuse should be avoided. It is necessary but can never substitute routine processing technique.

59. REFERENCES