Frozen Section: Principle

and Procedure 6

6.1 Introduction times, frozen section tissue is used for the

demonstration of fat and carbohydrate in the
The frozen section is the rapid tissue section by tissue sample.
cooling the tissue with the help of cryostat to give
immediate report of the tissue sample. This is
especially needed in large hospital to diagnose 6.3  he Principle of Frozen
T
the lesion or extent of the lesion at the time of Section
operation. The cryostat is the instrument that has
the arrangement to freeze the tissue and also to The rapid freezing of the tissue sample converts
cut the frozen tissue for microscopic section. the water into ice. The firm ice within the tissue
acts as embedding media to cut the tissue.
Lowering the temperature makes the tissue more
6.2 Indications of Frozen firm, whereas increasing temperature makes the
Sections tissue softer.
Cryostat Machine Proper (Fig. 6.1)
The frozen section is used mainly for immedi- Temperature range in the machine: The cryo-
ate diagnosis of the lesion for management and stat machine has the usual temperature range
to know the extent of the lesion [1–3] (Box from 0 °C to −35 °C. The most of the tissue is
6.1). It is also helpful to do enzyme immuno- sectioned properly between −15 °C and
chemistry and immunofluorescence study. At −25 °C. The water-containing tissues can be sec-
tioned in higher temperature, and fat-containing
tissue needs much lower temperature to cut.
Box 6.1 Indications of Frozen Section Rotary microtome (Fig. 6.2): Rotary micro-
• Rapid diagnosis of the lesion for intra- tome is placed inside the cabinet of the cryostat.
operative management Here the knife is fixed and the tissue is moved
• To know the extent of the lesion with the help of a rotary wheel.
• To do enzyme immunocytochemistry Tissue shelf (Fig. 6.2): Just in one side of the
• To do immunofluorescence study microtome, there is a tissue shelf to keep the tis-
• To stain lipid and certain carbohydrate sue. In this place the samples are kept for freez-
in the tissue ing. Usually the temperature of tissue shelf is
lower than the overall cabinet temperature.

© Springer Nature Singapore Pte Ltd. 2018 51

P. Dey, Basic and Advanced Laboratory Techniques in Histopathology and Cytology,
https://doi.org/10.1007/978-981-10-8252-8_6
52 6 Frozen Section: Principle and Procedure

Fig. 6.1 Cryostat

machine with its parts

within a metal frame. The undersurface of the

plate has free space, and there is a gap between
Rotary microtome
the knife and the plate.
Blade
Alternating to antiroll plate, a cool sable hair
Tissue shelf brush can be used to get unrolled tissue.
Antiroll plate Specimen holder: The specimen holder or
chuck is supplied by the manufacturers in differ-
ent sizes and shapes. Usually these are round
metal structures.
Embedding medium: This medium is used to
hold the tissue over the chuck. Presently opti-
mum cutting temperature (OCT) compound is
Fig. 6.2 Microtome, blade, antiroll plate and tissue
used as embedding medium. The OCT is made of
shelves are shown water-soluble glycols and resin.

Place to keep the brush and knife holder: Just 6.4 Cryostat Sectioning
in front of the microtome machine, there remains
a small place to keep the brush and knife holder. The process of the cryostat sectioning needs the
Knife or blade: Nowadays, low- or high-­ following steps.
profile disposable blades are used. The blade
should be proper fixed to the holder to get an even 1. Grossing and cutting the specimen (Box 6.2):
pressure in the whole length. Alternatively Profile The cutting surface of the tissue should be
C steel blade is also used. The angle of the knife smooth. The following steps in grossing of the
is kept in between 5° and 7°. tissue are mandatory for accurate reporting:
Antiroll plate (Fig. 6.2): Just in front of the • Identify the tissue sample of the patient and
knife, there is an antiroll plate that prevents the the requisition form: This is the first and
rolling of the cut tissue. It is usually a glass plate foremost part of the frozen tissue grossing.
6.4 Cryostat Sectioning 53

stroke of the scalpel rather than too much

Box 6.2 Grossing for Frozen Section Tissue pressure.
• Identify the tissue sample of the patient. Cytology of the tissue: At times the imprint of
• Clinical information: provides possible the tissue on the slide provides good morphologi-
differential diagnosis. cal details such as lymphoma of the lymph node.
• Tissue appearance: colour, texture, nod- Similarly crushing of tissue also provides excel-
ule, any suture. lent morphological details such as in case of tis-
• Anatomy of the tissue: identify the resec- sue of the brain tumour.
tion planes and margins. 1. Tissue embedding in the mould (Fig. 6.3): The
• Colour the resection planes and small piece of the tissue is kept in the centre of
margins. the mould, and then the OCT is poured over it
• Section cutting: in excess. Then the tissue holder or chuck is
–– Use sharp blade. firmly placed over the tissue with overflown
–– First cut the most important area. OCT.
–– Give gentle pressure and avoid too 2. Tissue loading in the frozen section chamber:
much pressure. The tissue is now placed in the frozen section
• Cytology preparation: if needed make chamber, and cold spray can be used to make
–– Imprint smear the process faster.
–– Scrape smear 3. Loading the blade: The cutting knife or blade is
–– Crushed smear now loaded and the proper alignment is done.
4. Trimming the tissue: The loss of normal or
natural colour to whitish colour indicates that
• Salient clinical information: The essential the tissue is frozen. The frozen tissue in the
clinical information is very helpful as it tissue holder is now placed in the holder of the
guides the pathologist to reach the possible microtome. The block is trimmed to remove
differential diagnosis. the excess OCT and to get the smooth tissue
• Tissue appearance: The gross appearance surface for sectioning.
of the tissue such as colour, texture, consis- 5. Sectioning (Fig. 6.3): The tissue is now cut
tency and any suture to mark the anatomi- gently and is spread over the antiroll plate
cal position. with the help of a brush. The brush should be
• Resection margin: It is very important to cooled. The tip of the tissue is guided by the
identify the resection margins of the brush.
tumour. The resection planes and margins 6. Section lifting: The glass slide of normal room
should be inked thoroughly. The different temperature is pressed firmly over the tissue
colours of ink can be used for medial and section, and normally the tissue sticks
lateral margin identification. immediately.
7. Fixation: The tissue should be immediately
Cutting the tissue: The tissue should be fresh fixed in methanol for 1 min or 95% ethanol for
without any fixative. The tissue should be prefer- few seconds. Rapid fixation within few sec-
ably dry, and it should not be wrapped in gauze onds is mandatory. In case of delayed fixation,
piece. Any suture, staple or sharp hard structure the cells are swollen, and the cytoplasmic
should be removed from the tissue sample. Now margin may be ruptured giving hazy appear-
the tissue is cut into small pieces as it facilitates ance of the margin of the cells.
freezing. Take multiple sections of the tissue to
understand the main pathology and to minimize Troubleshooting in frozen section: Various
the error. Use a new sharp scalpel blade, and first problems may arise during the cutting of frozen
cut the most important area that needs micro- section tissue. This has been highlighted in
scopic examination. It is preferable to use gentle Table 6.1.
54 6 Frozen Section: Principle and Procedure

a b c

d e f

Fig. 6.3 Cryostat processing: (a) mould is covered with cooling chamber, (e) the brush guides the tip of the tissue,
OCT, (b) the tissue is now put on the block, (c) OCT is (f) the tissue section is gently spread over the antiroll plate
flooded over the tissue, (d) the tissue now is put in the and later picked up by touching a glass slide

Table 6.1 Troubleshooting in frozen section

Problems Cause Solution
Freezing artefact Formation of ice crystal within the tissue. • Freeze the tissue rapidly, i.e. snap
Water-­containing tissue shows more such freezing
artefact • The tissue specimen should not be
in saline solution before freezing
Uneven tissue embedding The surface of the tissue is uneven, and • Make tissue even at the cutting
the vital information may be lost surface before freezing
Block is loosen during The chuck may be too cold when the • Take the tissue out and reattach it
chucking tissue is placed on it on a clean chuck which is not too cold
Tissue crumpled The tissue in the block is warm or too • Make the block of tissue in the
cold optimum temperature: −15 °C to
−20 °C
Chattering artefact The temperature of the block is too cold, • Bring the block in optimum
and the tissue becomes hard. The blade temperature. Pressing the cut surface
will cut the tissue thick and thin in of the block by gloved finger may
regular interval make the block warmer
Thin stripe in tissue The perpendicular tear in the tissue is due • Replace the blade by a sharper one
to nicks on the blade
Widely striped tissue and This may happen if the tissue is sticking • Clean the blade or replace with a
also tearing of the tissue with the blade new one

Fixation: Immediate dip in 95% ethyl alcohol 6.5.1 H&E Staining

for a few seconds fixes the tissue.
• Rinse the slide in tap water.
• Put in haematoxylin for 1 min.
6.5 Staining • Rinse in tap water for 5 s.
• Rinse in Scott’s tap water for 5 s for bluing.
We commonly use haematoxylin and eosin • Dip in eosin for 20 s.
(H&E) and toluidine blue stain. • Rapidly rinse in tap water.
References 55

• 95% ethanol for 10 s. Table 6.2 Optimum temperature for frozen section
• 100% ethanol for 10 s. Optimum
• 100% ethanol for 10 s. Tissue temperature
• Dip in xylene for 20 s. Brain, liver, spleen −7 °C to −10 °C
• Mount by DPX. Rectum, uterus, adrenal, muscle, −12 °C to −15 °C
skin
Heart, lung, intestine, pancreas, −16 °C to −20 °C
ovary, cervix, prostate
6.5.2 Toluidine Blue Stain Bone marrow, breast −20 °C to −25 °C

This is a very simple stain and takes only a few

seconds. The drops of toluidine blue stain are put on the knife and may make problem in
on the section, and the coverslip is put on the sec- cutting.
tion. The slide is now ready to see. The histopa- (ii) Collagenous tissue: The firm collagenous
thologist feels more comfortable in H&E stain tissue is difficult to cut.
than this unfamiliar toluidine blue stain. (iii) Necrotic tissue: Soft necrotic tissue may
create considerable problem as they may
fall from the slide making hole in the sec-
6.6 Factors Affecting the Good-­ tion. It is preferable to take only viable
Quality Section tissue for frozen section.
(iv) Bony hard tissue: Hard tissue like bone
The common factors responsible for the good-­ or cartilage may damage the blade sig-
quality smear include: nificantly. In this situation a new section
can be processed, or new blade can be
• Temperature: when the temperature falls, used.
water within the tissue becomes frozen and • Tissue size: The size of the tissue sample
gives the tissue hard consistency. The opti- should be small as the larger tissue takes much
mal temperature of frozen tissue is in longer time to freeze.
between −15 °C and −25 °C. Warm tissue
remains soft and sections crumple. On the
other hand, the overcooled tissue becomes References
very hard and brittle and produces again
bad-quality crumpled section. Moreover the 1. Ayhan A, Ozler A, Dursun P, Haberal AN. Potential
role of increasing number of sections in frozen sec-
hard tissue may cause “chattering” artefact tion diagnosis of ovarian tumors. J Exp Ther Oncol.
and also thick and thin sections. Different 2016;11(4):245–50.
tissue contains variable amount of fat and 2. Chambers KJ, Kraft S, Emerick K. Evaluation of fro-
water. The consistency of different tissue zen section margins in high-risk cutaneous squamous
cell carcinomas of the head and neck. Laryngoscope.
varies, and therefore the optimum tempera- 2015;125(3):636–9.
ture to cool the tissue varies considerably. 3. Hatami H, Mohsenifar Z, Alavi SN. The diagnostic
Table 6.2 shows the optimum temperature accuracy of frozen section compared to permanent
of different organs to have good frozen section: a single center study in Iran. Iran J Pathol.
2015;10(4):295–9.
section.
• Tissue consistency: other than the optimum
cooling temperature, the consistency of tissue
has significant effect on cutting such as:
(i) Fatty tissue: It is difficult to cut the fatty
tissue in frozen section. Fat may smear