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Frozen section
- 2. ● The frozen section procedure is a pathological laboratory procedure to perform rapid microscopic analysis of a specimen. ● The technical name is CRYO-SECTION. ● It is a method that produces the sections without dehydrating, clearing agents and embedding media as well.
- 3. ● History – Resulted from need for rapid intraoperative Diagnosis. – Mayo clinic chief of pathology Louis B Wilson pioneered the frozen section in 1905.
- 4. ● Uses of Frozen section in histology laboratory – Rapid production of sections for intraoperative diagnosis. – Diagnostic and research of enzyme histochemistry. Enzymes are labile – Immunofluorescent methods – Immunohistochemistry methods: heat and fixation may inactivate or destroy antigens. – Diagnostic and research non-enzyme histochemistry – eg lipids and carbohydrates. – Silver methods used particularly in neuropathology.
- 5. ● Indications of frozen sections. – Urgent diagnosis during surgery – Determine the involvement of resection margins in malignancy. Eg Basal cell carcinoma – Identifying ganglion cells in Hirschprung disease. – Enzyme histochemistry (ATPase, NADPH) – Non-enzyme histochemistry (lipid, carbohydrate) – For silver and gold impregnation methods – Identifying type of tissue eg parathyroid.
- 6. ● Theoretical considerations – Principle of frozen sections ● When the tissue is frozen, the water in the tissues turns to ice. The tissue becomes firm in this state. ● The ice acts as the embedding media. ● The consistency of the frozen block can be altered by altering the temperature of the tissue. Decreasing the temperature produces harder block while increasing temperature softens the block. ● Majority of non-fatty unfixed tissue section well at -25 deg C. ● Sectioning of fixed tissue requires a block at -10 deg C.
- 7. ● Fixation – Frozen section requires unfixed tissues. – Post fixation – for enzyme histochemistry (controlled conditions)
- 8. ● Freezing Techniques – Liquid nitrogen ( -190 deg C) – Isopentane cooled by liquid nitrogen ( - 150 deg C) – Dry ice ( -70 deg C) – Carbon dioxide ( -70 deg C) – Aerosol sprays ( - 50 deg C)
- 10. ● Methods for suitable freezing – Cryostat ● Microtome is inside the chamber ● Microtome is temperature controlled – Freezing microtome ● Tissue fixed separately ● Microtome is not under temperature control.
- 11. Cryostat ● The cryostat is a refrigerated cabinet in which a specialty microtome is housed. ● All the controls to the microtome is operated outside the cabinet. ● Features – Electronic temperature control – Electronically controlled advance and retraction of the block. – Specimen orientation facility
- 13. – Digital visualization of chuck and cabinet temperature. – Mechanical cutting speed control and section thickness – Automatic defrost mechanism – Automatic de-contamination and sterilization.
- 14. ● Types of cryostat – Closed-cycle cryostat – Continuous-flow cryostat – Bath cryostat – Multistage cryostat
- 15. ● The best quality frozen sections are produced from fresh unfixed sections that has been rapidly frozen. ● Cryostat freezing may produce freezing artifact because of slower freezing than other techniques. ● The water in the tissues form ice crystal when the tissue freezes.
- 16. ● The size and quality of the crystals is proportional to the speed at which the tissue is frozen. ● After cutting, the sections are placed on a room temp slide → thawing of the tissue → produces freezing artifact appearing as holes in the tissue when viewed microscopically. ● Method of choice of freezing. – Isopentane and – liquid nitrogen (problem : produces vapor bubbles around the tissue → acts as insulator → prevents rapid cooling. → freeze artifact.
- 17. ● To localize hydrolytic enzymes eg acid and alkaline phosphatase, and other antigens, the tissue is fixed prior to freezing and sectioning. ● Method – Tissue must be fresh – Place in formal calcium at 4 deg C for 18 hours for fixation. – Rinse in running water or Distilled water(if the tissue is fragile)
- 18. – Blot dry – Place tissue in gum sucrose solution for 18 hrs at 4 deg C – Blot dry – Freeze tissue onto block holder
- 19. ● Cryostat sectioning – Cabinet temperature ● Should be suitable for tissue type and type of preparation to be cut ● Most unfixed material : -15 deg C to -23 deg C ● Tissue containing large amount of water or fixed tissues section best at warmer temperature. ( -7 to -12 deg C) ● Tissue containing fat require a colder temperature. ● Shattered sections – block is too cold.
- 20. ● Microtome – In case of cutting problems – defrost microtome, and oil as recommended. ● Blade/knife – Disposable blades have replaced stainless steel knives. – Type of tissue and procedure to be performed dictate the use of knives. – Sharpening techniques should be mentioned in procedure manual. – Sharp edge is essential for proper section.
- 21. ● Disposable blades have perfect edge and instantly available. ● They have an advantage of sharpness, and the blades are rapidly cooled due to their size. ● Hard or dense tissue may be troublesome for disposable blades.
- 22. ● Anti-roll plate – Piece of equipment attached to the front of microtome. – Prevents the upward curling of frozen section during sectioning. – Made of plexiglass or hard plastic. – Aligned parallel to the blade and slightly above it.
- 23. ● Sectioning technique – Speed, tissue type, and temperature of the block and the cabinet is vital in sectioning. – After cutting, the cut section will rest on the blade holder. – Room temperature slide is held above the section. Electrostatic attraction causes the tissue to adhere to the slide. – If staining technique to be used is lengthy, positively charged slides should be used.
- 24. ● Coatings – Gelatin – formladehyde mixture ● Gelatin 1% (5ml) ● Formaldehyde 2% (5ml) ● Coat slides, allow to dry at 37 deg C for 1 hr or overnight before picking up sections. – Poly – L – lysin coating (0.01% PLL aqueous) ● Wash slides in detergent – 30 min ● Wash slides in running tap water – 30 min ● Rinse slides in DW two times – 5 min each
- 25. ● Wash slides in 95% alcohol twice – 5 min each ● Air dry slides – 10 min ● Smear 20 micro liter of PLL over each slide ● Air dry and store dust free This technique is used as section adhesive in immunohistochemistry.
- 26. ● RAPID BIOPSY FOR INTRAOPERATIVE DIAGNOSIS – Frozen section is valuable for rapid diagnosis during surgery. – Selected tissue is frozen using one of the freezing techniques. – Slide is immediately immersed in cold acetone or 95% alcohol. – Sections are immediately stained by rapid H&E, methylene blue, or polychrome stain.
- 27. ● Ultracryotomy – Used in research laboratory. – Rapid freezing of fixed or unfixed tissue using isopentane or liquid nitrogen. – Sections cut at 50 -150 nm.
- 28. ● Freeze drying and freeze substitution. – Freeze drying is a technique of rapid freezing (quenching) at -160 deg C and subsequent removal of water molecules by sublimation in a vacuum at a higher temperature (-40 deg C). – The blocks are raised to room temperature and fixed by vapor. – Advantages – this technique minimizes : ● Loss of soluble substances.
- 29. ● Displacement of cell constituents. ● Chemical alteration of reactive groups ● Denaturation of proteins ● Destruction or inactivation of enzymes.
- 30. Thank You