Frozen Section Basics

Frozen Section basics are described here. Courtesy to BSMMU, Dept of Pathology.

2. Frozen
Section
Presented By:
Dr. Nafisa Abedin
MD, Pathology,Resident Phase-A
;Dept. of Pathology, BSMMU.
Supervised by
Dr. Ferdousy Begum
Associate Professor
Department of Pathology,
BSMMU.

3. What is frozen section?
The frozen section procedure is a pathological
laboratory procedure to perform rapid microscopic
analysis of a specimen. The technical name for this
procedure is Cryo section

4. History:
Sir Louis B. Wilson
Frozen Section Resulted From
Need for Rapid Intra operative
Diagnosis.
Mayo Clinic Chief of Pathology
Louis B. Wilson pioneered the
frozen section at the Rochester,
Minnesota clinic in 1905 .

5. Frozen section–indications
1.Urgent diagnosis during surgery ( benign and
malignant)
2. Involvement of resection margins by malignancy, eg
basal cell carcinomas
3.Ganglion cells in Hirschprung disease
4. Enzyme histochemistry (ATPase, NADPH in muscle
biopsy samples)
5.Non enzyme histochemistry (lipid,carbohydrate)
6. For DIF, silver and gold impregnation methods
7. Identification of the type of tissue, eg. parathyroid
gland

6. What pathologist should know before
frozen section:
 
1)Cryostat machine
2)Request form
3)What surgeon wants to know?
4)what surgeons have sent?
5)How to contact them?

7. Request form for frozen
section,Pathology
department ,BSMMU

8. What pathologist should know during
frozen section
1)Whether tissue selection is appropriate or not
2) Type of tissue should cut at proper temperature.
3)Time consideration(About 20-30 min)
4) Communication with surgeon.

9. Flow chart of standard
operating procedure at BSMMU

10. Grossing station of BSMMU

11. Personal protection

12. Section of the tissue:
1)Tissue sample should represent the specimen
2)should not contain any necrotic area
3)3-4mm is ideal section
4)Sample once collected should be frozen immediately

13. Fixation:
Frozen section procedure requires unfixed tissue
Post fixation: for enzyme histochemistry(in controlled
condition)

14. Principle
Tissue
is
Frozen
Water in

Tissue
Turns to
Ice
Tissue
Becomes firm
And water
Act as
embedding
medium

15. Freezing techniques:
•Liquid nitrogen (-190
0
c)
•isopentane cooled by liquid nitrogen (-150
0
c)
•dry ice(-70
0
c)
•carbon dioxide gas(-70
0
c)
•aerosol sprays(-50
0
c)

16. Ideal temperature chart

17. Methods for suitable freezing
1)cryostat:
-microtome is inside the chamber
-microtome in under constant temperature control
2)Freezing microtome:
-Tissue fixed separately
-Microtome is not under temperature control

18. Cryostat Machine

19. Cryostat:
Types:
1)Closed-cycle cryostats
2)continuous-flow cryostat
3)Bath cryostat
4)Multistage cryostats

20. Cryostat chamber
Inside cryostat chamber
Inside cryostat chamber

21. Fig: Various electronically controlled parts of cryostat

22. Operation of Cryostat
Sharp blade

23. Points should be remembered while
cryostat sectioning:
•Orientation of the tissue
•cutting surface
•embedding medium.
•Position of the tissue to the blade
•The optimum cutting temperature

24. Cryostat sectioning technique
Using the brush
Using the anti
role plate

25. Frozen section embedding medium

26. Taking of sections from stage
The cut section will rest on the surface of
the blade holder after cutting ,a room
temperature slide is held above the section,
where electrostatic attraction causes the
tissue to adhere to the slide.

27. Staining:
1)Rapid H-E method
2)Toludine method
3)Methylene Blue
4)Methyl violet for amyloid
5)Oil red O for fat
6)PAS
Fig: Different stains

28. Technique of staining
•Immediately fix frozen section in 95% ethyl alchohol
•Rinse well in distilled water
•Stain with Hematoxylin Stain
•Wash well in distilled water
•Counterstain in Eosin Y Working Solution
•Dehydrate in ethyl alcohol
•Subsequent treatment

29. Pros and cons of frozen section
Pros:
•Fastest of all methods
•Excellent for IHC, IF, ISH.
•Often easiest to section-depending upon the tissue.
Cons:
•Poorest morphology
•Prone to freezing artifact-must be snap frozen

30. Frozen section Vs routine section:
Poor morphology in frozen section in comparison to routine section

31. Common problems: artifacts
•Ice crystal artifacts:
- Cause: slow freezing of tissue
- Solution:Freeze fast(flash/snap)
- It is a chemical property of
water, that water will expand on
freezing.
Fig: Ice crystal artifacts (Clefts
and vacuoles in the specimen)

32. Common problems: artifacts
•Knife artefacts

33. Common problems: over freezing
•Over freezing can cause
sections to have holes.
Tissues with high water
content eg. edematous or
bloody tissue will show this
problem.
Kidney tumor is a good example
of water content causing this
problem

34. 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
•Unlabeled slides should never be used

35. Quality assurance
•Correlation has to be done in each case.
•The cryostat must be clean periodically.
•Less frequently used machine can be decontaminated
on a longer cycle
•Records keeping.

37. •Sometimes the procedure is misused by surgeons to
satisfy their curiosity
•To compensate their deficiency in recognize normal
anatomical structure.
•To communicate the results immediately to the
patient’s relative.
Misuse of frozen section

38. Take home message
•Definitive diagnosis is not possible in all cases.
•Important for surgeons to influence the mode of
surgery
•Misuse must be avoided.
•It is necessary but never substitutes routine tissue
processing.