FROZEN SECTION

Dr Snigdha Sinha

Lesson Plan:
History
Introduction
Principle
Uses
Procedure
Technique
Advantages
Limitations

History:
The frozen section procedure as practiced

today in medical laboratories is based on the

description by DrLouis B. Wilson in 1905.

Introduction
Frozen section is a specimen of tissue that

has been quick-frozen, cut by microtome, and

stained immediately for rapid diagnosis of
possible malignant lesions
The technical name for this procedure
iscryosection

Theintraoperative consultation-

intervention by thepathologist, includes

Frozen section
Gross evaluation
cytology preparations (e.g. touch imprints)
special studies (e.g. molecular pathology
techniques, flow cytometry).

Principle

When the issue is frozen the water within the

tissue turns to ice -hence the tissue becomes

firm- the ice acting as the embedding medium.

Uses
1)Rapid production of sections for intra operative
diagnosis
2)In the performance ofMohs surgery Surgery for skin cancers
Intraoperatively -After each removal of tissue
-tissue is examined for cancer cells- surgeon is
informed so that additional tissue can be
removed.
Uses- basal cell carcinoma , squamous cell
carcinoma

Contd..
3)If a tumor has metastasized -sample of the
suspected metastasis sent for cryosection .
Aggressive surgery is performed if there is a
chance to cure the patient.
If the tumor has metastasized, surgery is not
curative- surgeon will choose a more
conservative surgery, or no resection at all.

Contd..

4)After a tumor is resected to check if surgical

margins are free of tumor- to assess the need
to do further resection for clearing margins
5)In asentinel node procedure - a sentinel node
containing tumor tissue prompts a further
lymph node dissection, while a benign node will
avoid such a procedure.
6)If surgery is explorative-rapid examination of a
lesion to identify the possible cause of a
patient's symptoms- followed by further
intervention.

Contd..

7) Immunofluorescence methodology
8) Immunohistochemistry techniques- when heat
and fixation may inactivate or destroy the
antigens
9)Diagnostic and research non-enzyme
histochemistry, e.g. lipids ,carbohydrates

Contd..

10)silver demonstration methods- particularly in

neuropathology
11)diagnostic and research enzyme
histochemistry
for labile enzymes

Procedure

key instrument for

cryosection -cryostat
-microtome inside a freezer.
Sections cut at 5 - 10
micrometres.

The surgical specimen is placed on a metal

tissue disc which is then secured in a chuck and

frozen rapidly to about 20 to 30C.

Contd..

The specimen is embedded in a gel like

medium calledOCT(optimal cutting

temperature compound) consisting of
polyethylene glycol andpolyvinyl alcohol when frozen has the same density as frozen
tissue.

Contd..
Usually a lower temperature

is required for fat or lipid rich

tissue. Each tissue has a
preferred temperature for
processing.
Subsequently it is cut frozen
with the microtome portion of
the cryostat, the section is
picked up on a glass slide and
stained (usually with
hematoxylin and eosin.

Freezing methods:

The best-quality frozen sections are produced

from fresh unfixed tissue which has been

rapidly frozen by1) Liquid nitrogen (190C)
2) Isopentane -cooled by liquid nitrogen
(150C)
3) Dry ice (70C)
4) Carbon dioxide gas (70C)
5)Aerosol sprays (50C)

The best frozen sections are obtained when

the tissue is frozen very quickly.

The method of choice - isopentane and liquid
nitrogen.

Technique
1)Grossing the tissue All the surfaces to be examined noting- color,
texture, consistency, nodules, defects,
adherent tissues, sutures, and any deviations
from normal anatomy
Resected margins to be examined for staging
and therapeutic outcome

Contd..

Inking the resected margins- to check for

invasion
Black india ink is commonly used
If tumor is extending to the inked surface its
reported as a positive margin surgeon will
excise more tissue from the site to ensure a
complete excision of the tumor

2)Proper Communication with the SurgeonsThis means knowing what tissues have been
removed previously,
Reviewing any previous diagnosis or slides on
the patient- because the present procedure
may be related to previous ones.

3) Embedding the tissue:

Tissue is placed face up on the
chuck and covered with
embedding medium(OCT) The
chuck is placed on a designated
freezing surface in the cryostat.
After a period of partial freezing,
a weighted heat extractor is
placed on the top of the chuck to
flatten the tissue surface to
complete the freezing process
4- Cryostat:
Tissue is cut with the microtome
portion of the cryostat by setting
the thickness to 10microns.

a) Temperature:
The temperature should be at -20F
for most tissues. For tissues with a
large fat component, -40F is
needed.
Too high, i.e., -10F - tissue will not
stay frozen and firm. Too cold, i.e.,
-50F and the tissue will crumble
and become powder.
b) Blade sharpness and angle:
The blade should be sharp ,should
be changed once every 2 weeks ,
angle between the blade and the
plane of movement of tissue should
be optimal(2-4deg-paraffin,
5-7deg-frozen)
Once the tissue is cut a slide is
placed face down and the tissue is
picked up and stained

5. Staining:

Once the tissue is on the slide it can be

either air-dried or fixed in methanol. This
depends on which staining procedure will
be used (delay can cause artifacts)
Steps Formalin or 95% alcohol: 45-60
seconds
Water: 5-7 seconds
Hematoxylin: 60 seconds
0.2 % aqueous ammonia (Bluing): 1520 seconds
Eosin: 20-60 seconds
95% alcohol: 10 seconds
100% alcohol: 10 seconds
Xylene, Toluene: 10 seconds
Mounting media for cover slipping
Whole procedure taking about 4minutes

Advantage

Disadvantage

H&E

Looks like permanent

section staining

Takes 3 minutes

Toluidine Blue

Takes 10-20 seconds

Different appearance
than
permanents

Giemsa

Takes 10-20 seconds

Different appearance
than
permanents

Contd..
6) Interpreting The Frozen SectionThe report is limited to a "benign" or
"malignant" diagnosis, and communicated to
the surgeon operating via intercom.

Advantages:

1) The preparation of the sample is much more

rapid than with traditional histology technique
(around 10 minutes vs 16 hours).
2)If more tissue is needed to make an accurate
diagnosis, the surgeon is able to obtain an
additional sample, avoiding a second
operation.
3)If the tissue is determined to be cancerous- the
mass can be removed at that time.

Contd..
4)If the tissue is determined to be benign
surgery can end.
5)Ensures that the mass being removed is the
intended tissue for removal.
6)Ensures that the entire mass and its
surrounding borders are removed.
7)It allows for the collection of proper tissue
samples for further scientific research.
8)The surgeon and pathologist are able to
collaborate to care for the patient

Limitations

1)Freezing artifact- edematous tissues freeze with an

appearance similar to soap bubbles- as the water
freezes-expanding water- forms rounded ice crystals which
compresses the strands of fibrous tissue giving appearance
of bubbles.
Edematous tissues can difficult to cut without shattering due
to the icy consistency
picture on the left shows freeze artifact in the stroma and the
picture on the right shows edematous nature of stroma

2)Compression Artifacts-tissues will be

compressed by expanding ice bubbles. This is
most evident in edematous tissues
The picture on the left shows the renal tubules
being compressed by the clear ice crystals. The
picture on the right shows tissue which was not
frozen.

3)Nuclear ice crystals

Nuclei will show varying tendency to form ice crystals.
Vesicular nuclei has greater tendency to show ice crystals.
4) Thickness
The thinner the tissue is cut - more these crystals appear as holes
Tissue on left is cut at 6 micron , tissue on right is cut at
3micron(uterine sarcoma)

Contd..
5)Drying artifacts -the nuclei of a frozen
section left for more than few seconds to dry
show a loss of definition.
6)Sampling error- observation, examination ,
inking and grossing to be done in a meticulous
manner.
7)Fat-difficult to freeze.
8)Inconsistency of training and
performance- for carrying out the procedure
can prove a limitation.

Because of the poor technical quality of the

frozen sections- A final diagnosis is rarely

offered intraoperativel y