Histopathology

1. M R . M A H M O U D I B R A H I M
FROZEN SECTION AND
CRYOSTAT

4. INTRODUCTION
•Frozen sections are methods used to
produce sections without the use of
dehydrating and clearing solutions, and
without embedding media.

5. •The preparation of the sample is much
more rapid than with traditional histology
technique (around 10 minutes vs 16
hours). However, the technical quality of
the sections is much lower.

6. •The principle of frozen section is
converting the water within the tissue into
ice which act as embedding media to
enable to cut thin section to establish
rapid diagnosis.

7. •There are two type of frozen section:
•1 Fresh unfixed frozen section (most
common)
•2 fixed frozen section.
•Cold formal calcium 4 C for 18 hrs.

8. •Frozen sections have important clinical
and research applications. Clinically the
use of frozen sections for intra-operative
consultation.

9. USES OF FROZEN SECTIONS
•The production of frozen sections has
many applications in routine histology
laboratories:
1-Rapid production of sections for intra-
operative diagnosis

10. 2-Diagnostic and research enzyme
histochemistry or labile enzymes
3-Immunofluorescent methodology
4-Immunohistochemistry techniques when
heat and fixation may inactivate or destroy
the antigens

11. 5-Diagnostic and research non-enzyme
histochemistry, e.g. lipids and some
carbohydrates
6-Sliver demonstration methods,
particularly in neuropathology

12. METHODS OF FREEZING
TISSUE
•1-liquid nitrogen -190.
•isopentane (2-methylbutane) cooled by
liquid nitrogen (−150°C)
•3-dry ice (−70°C)

13. •4- carbon dioxide gas (−70°C)
5- aerosol sprays (−50°C)

14. ADVANTAGES
•Rapid, simple and easy to reproduce
•Disadvantages:
•Loss of cellular details
• more difficult to obtain serial sections
from the same specimen.

15. •Refrozen of specimen can damage the
tissue.
• Staining is not very good
• Some specials stains cannot be
performed
•Lack of consultation

16. THEORETICAL CONSIDERATIONS
•The principle of cutting frozen sections is
simple: when the tissue is frozen, the
interstitial water in the tissue turns to ice,
and in this state the tissue is firm with the
ice acting as the embedding medium.,

17. •The consistency of the frozen block may
be altered by varying the temperature of
the tissue. Reducing the temperature will
produce a harder block; raising the
temperature makes the tissue softer.

18. •The majority of non-fatty unfixed tissues
section well at −25°C. The sectioning of
fixed tissue requires a block temperature
of approximately −10°C or warmer.

19. PREPARING TISSUE FOR FREEZING
•Tissue for freezing should be frozen or
fixed as promptly as possible after
cessation of circulation to avoid
morphological distortions and damage
due to:

20. •Tissue drying artifact.
• Autolysis - The destruction of tissues or
cells by the action of substances, such as
enzymes, that are produced within the
organism. Also called self-digestion.

21. FREEZE DRYING TECHNIQUE
•Little used in routine
•Used in immunohistochemistry
•The technique minimizes the:
•Loss of soluble substances

22. •Displacement of cell constituents
•Chemical changes of reactive group

23. •Principle
•1-Rapid freezing of fresh tissue -160C
(quenching) then removal of water in
form of ice by sublimation at high
temperature -40 C

24. •The freeze dried blocks are then raised to
room temperature and either fixed by
vapor fixative or embedded in a suitable
medium

25. •Freeze -drying can be considered in four
stages:- a. Quenching b. Drying c.
Fixation & embedding d. Subsequent
treatment

26. APPLICATION OF FREEZE DRYING
•Demonstration of hydrolytic enzyme
•Fluorescent Antibody studies
•Mucosubstance
•Proteins
•Scanning EM

27. FREEZE SUBSTITUTION
•Alternative method ,easy and rapid
method good for enzyme,protiens and
mucosubstance preservation of lipid is
poor.

28. •This technique involves the rapid
freezing of small pieces of tissues in a
similar manner as for freeze drying
Substitution

29. •of the ice in such tissue by placing them
in dehydrating agent at sub zero
temperature.

30. CRYOSTAT
•Cryostat is a device by which temperature
can be maintained in a low level.
•In pathology and histology it is known as
chamber containing a microtome for
sectioning frozen tissue

31. Electronic temperature control.
The temperature can be depending on the
tissue being cut usually between -20 to -30
C

32. OPTIMAL CRYOSTAT CUTTING
TEMPERATURES
FOR UNFIXED TISSUES:
•Brain, lymph node,liver,kidney,spleen
And testis at -12 to -16 C.
•Breast,skin,thyroid,adrenal,muscles and
prostate at -18 to -30 C

33. •Soft tissues cut better at slow rate while
the hard tissues better at slightly faster
rate.
-Fixed tissues is more difficult to cut
well.

34. •Cutting section pick up on slides or
cover-slips,the 40C difference in
temperature between section & slide
enough to adhere the section to slide

35. •Fixed section have a tendency to detach
during staining , to avoid this coat the
slide in gelatine-formaldehyde mixture,
or poly-l-lysine