Frozen section

technique of frozen seciton

1. 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