The main reason to use this technique and all stepes to achieve perfect slides to get right diagnosis.

1. Frozen section and cryostat
Presented by
Dr. HADEEL. Khalaf.
ALBOAKLAH
Dr. Muntader Mohammed
Kani

2. FROZEN SECTION :
 A frozen section is conducted during surgery on patients
with ovarian and pelvic masses while patients are under
anaesthetic. While patients are asleep, the surgeon
removes tissue from a suspicious mass from an ovary or a
uterus, and sends it to a pathologist who is present in the
operating theatre. The pathologist freezes the tissue with
liquid nitrogen, cuts it, and then stains it with special
staining solutions so that it can be viewed under a
microscope.

3. The principles of frozen sections

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

5. Type of frozen section
There are two type of frozen section:
1. Fresh unfixed frozen section (most
common)
2 fixed frozen section.
Cold formal calcium 4 0C for 18 hrs.
Frozen sections have important clinical
and research applications. Clinically the
use of frozen sections for intra-operative
consultation.

6. The production of frozen sections has
many applications in routine histology
laboratories:
1-Rapid production of sections for intra-operative.
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.
5-Diagnostic and research non-enzyme
histochemistry, e.g. lipids and some
Carbohydrates.

7. METHODS OF FREEZING
TISSUE
1-liquid nitrogen -190.
2.isopentane (2-methylbutane) cooled by
liquid nitrogen (−150°C)
3.dry ice (−70°C)
4- carbon dioxide gas (−70°C)
5- aerosol sprays (−50°C)

8. Advantage &disadvantages:
 Rapid, simple and easy to reproduce
 Disadvantages:
 Loss of cellular details
 more difficult to obtain serial sections from the same specimen.
 Refrozen of specimen can damage the tissue.
 Staining is not very good
 Some specials stains cannot be performed
 Lack of consultation
Frozen section of
lungs
Normal section of
lungs

9. Rapid fixation
• As I mentioned earlier I hold the slide in my right hand as I turn the wheel. The
moment the section is complete, I immediately pick up the tissue on the slide and in a
moment it is placed into fixative.
• Have your fixative opened in an immediately reachable location. Start with the slide in
your hand. If am using a staining rack I keep it outside the fixative jar so it does not
impede my swift motion. I first fix the slide in 95% ETOH then put it in the rack
• If there is delay in fixing the tissue there will be significant drying artifact. In my
experience when the frozen section is sitting cold on the stage the effect of drying is
minimal. From the time the tissue touches a warm slide it starts to under go
significant drying artifact with loss of nuclear detail and leakage of fluids from the
cytoplasm.

10. Rapid fixation
 The examples below show the same tissue after 15 seconds delay of fixation
on a warm slide and sections fixed immediately . The differences are striking.
It also demonstrates the quality of cytology possible by frozen section using
this system.
Bronchiolo-alveolar
Carcinoma - 15 seconds
drying
Same tissue
immediately
fixed 95% ETOH

11. Retrieving from the block
 1) A section is cut leaving
an attachment of
medium at the top
 2) The wheel is turned in
opposite direction bring
the section back to the
face of the block.
 3) Section is retrieved by
placing the slide over the
tissue on the face of the
block.

12. Retrieving from the block

13. Thickness of the section
 For general surgical pathology I recommend cutting at six microns. This
thickness will give a
 rich stain which is easier to interpret at scanning powers of 2x and 4x where
pathologists gather much of their information. Very thin sections will often
look pale at these powers and fine details are easy to miss.
 A six micron section will afford a moment more time to avoid drying artifact.
There will also be less nuclear “holes” visible from ice crystal artifact.
 Specialized situations may call for thinner or thicker sections.

14. Staining the sections
 Individual staining recipes are a matter of pathologist preference
 keeping the staining uniform, but the type of tissue and its adhesive nature
should be considered.
 Get to know the colour and shade of a well stained slide as compared to a
lightly stained slide.
 Keep in mind the thickness of the tissue and the amount of nuclear material
will make the slide appear lighter or darker. However the is a particular tone
of blue that tells me the slide is well stained. Make your own observations.
The idea is to check the slide before continuing on with the staining.

15. Staining the sections
 When looking at lymph nodes for metastatic disease I give particular attention to
deep rich staining, especially the eosin. If the eosin stain is rich, the the pale pink
cytoplasm of a sinus histiocytic can be more easily distinguished from the tumor
cell cytoplasm which may be more eosinophilic or clear more clear. I can list
several situations where I have experienced sections coming off the slide in the
staining process.
 1) Very dry tissues either by nature or desiccation. "Less glue"
 2) Large ratio of border to area. Thin strips that have a large border to catch the
disorder of the motion in the stain jars can easily fall off the slide. This is
especially true if thin fibrous walled cysts which are not very "juicy" tissues to
begin with. Also includes in this are amorphous necrotic tissues which have no
integrity holding them together. This also applies to very thick sections which
have a thicker wall to grab the turbulence.

16. Staining the sections
 3) Ammonia bluing reagent is too concentrated.-If you use ammonia for bluing
my rule is if I can smell it without putting my nose up too it its too strong.
 4) 100 % Etoh instead of 95%. I have on occasion had someone place 100% Etoh
in my fixing jar instead of 95%. In my experience tissue will not stay on the
slide.
 5) A section is placed over embedding medium which is already on the slide.
When placing multiple sections on a slide be careful to not overlap the tissue
onto the embedding medium of the neighbouring section.

17. Frozen Tissue Slide Preparation and
Processing