FROZEN SECTIONING & CRYOSTAT

 FROZEN SECTIONING

Aim/Significance

1. For certain staining procedures, e.g. the demonstration of lipid by Oil red O method,

for certain impregnation methods, e.g. the silver impregnation, frozen sections are

essential.

2. Frozen sections are indispensable for rapid diagnosis of malignancies during

operations, e.g. metastatic tumors of breasts and lymphomas of different types. In

such cases time matters and quick reporting is required.

3. All enzymes are destroyed at temperature above 56°C and they all are best shown in

cryostat or frozen sections of fresh tissue. Although, phosphatases may be

demonstrated in paraffin section.

4. Frozen sections are thicker and easily be handled during staining. 

5. Frozen sections are also helpful in immunohistochemistry where antigen antibody

complexes are studied in tissues as in lymphomas.

Disadvantages

1. Cost Effect:

To produce frozen sections, special devices like cryostat are required. These

instruments are very expensive and routine laboratories cannot afford it.

2. Poor Staining:

Staining on frozen sections is rarely satisfactory. The colours are usually faint.

3. Freezing Artifact:

Freezing artifact may be produced by inappropriate techniques, e.g. 

x Presence of ice crystals in tissues;

x Nuclear ballooning and vaculation;

x Separation of a mucosal or other epithelial surface.

4. Poor Quality Sections:

It is almost impossible to obtain serial sections on frozen section.

5. Because of the lack of an embedding mass, structural details tend to be somewhat

distorted during cutting and handling.

6. For frozen sections a lot of skill, experience and supervision is required.

7. Different tissues have different optimal cutting temperature (O.C.T) and it is very

difficult to acquire temperature changes for different types of tissues.

O.C.T. for Different Tissues

I II II

-18 to -20 °C

Lymphnode, soft

tumors,

endometrial

curvettings

-15 to -25 °C

Uterus, cervix, nonfatty skin, non-fatty

breast

-35 °C

Fatty skin, fatty

breast, omentium

tissue

Principle of Frozen Sections

The principle of frozen sections is very simple. As a tissue is frozen, water present in it is

converted to ice which then gives an internal support to the tissue and its sectioning is

facilitated. The ice acts as an embedding medium. Frozen sections can be produced from

fresh as well as fixed biopsy. But the former is preferred than fixed material but it is

hazardous so UV light is used for disinfection.

When quick reporting is required frozen sections are taken from fresh material because no

time for fixation is available. The use of fresh material is associated with problems like risk

of infection and streaming artifact. On the other hand, fixed tissues can be used when the

aim is histochemical study. Fresh biopsy material can be easily sectioned in a cryostat

because they require O.C.T in the range of 0 to -15 °C to -30 °C which is easy to obtain in a

cryostat. On the other hand, fixed biopsy material cannot be excellently sectioned in a

cryostat because they require an O.C.T which is difficult to attain on a cryostat.

Freezing microtome is commonly used for fixed frozen sections because O.C.T for fixed

biopsies can easily be produced which is not possible for fresh biopsy material. The fact

that fixed biopsies require a higher O.C.T than that for fresh biopsy which requires a lower

O.C.T is due to difference in their water content. Fixed biopsy contains more water and so

are firmly solidified, even at a higher temperature -15 °C to -10 °C whereas fresh biopsies

are firmly solidified at a much lower temperature like -15 °C to downwards.

The working principle, merits and demerits of freezing Microtome are described in chapter
6.

CRYOSTAT

The best method of preparing sections from unfixed tissues is by use of a cryostat. The

cryostat consists of a microtone housed in a deep freeze cabinet, maintained at a

temperature of -15°C to -30°C. A thermostat controls the temperature of cabinet. The

optimum working temperature of cryostat is -18°C to -20°C. The mounting media used in

cryostat is O.C.T which stands for optimal cutting temperature. The microtome is handled

by an operating handle outside the cabinet. There is various cryostats fabricated, the

fundamental difference between them is the type of microtone employed. The earliest

models manufactured in UK incorporated the Cambridge Racking microtone. However, 

now a days rotary microtome is commonly used with wedge profile knife because this type

of knife is very rigid. Several models of cryostat fitted with Base sledge microtone, suitable

for cutting larger and tougher tissue blocks are also available.

Anti Roll Plate

In cryostats a special device called Anti Roll Plate is incorporated which is meant for

prevention of curling or rolling of frozen sections. This device is adjusted to knife edge in

such a way that a small gape or slot is present between knife edge and anti-roll plate. As

frozen sections are produced these smoothly go beneath the anti-roll plate on the surface of

the knife. Anti roll plate is necessary because frozen sections show a natural tendency of

curling.

When sections are produced, anti-roll plate is set apart and sections are directly picked on

slides, properly smeared with an adhesive and kept at room temperature. As there is

temperature difference between sections (-20°C) and slides (room temp) so due to this

difference in temperature sections firmly stick with the slide. Frozen sections of fresh

material rarely detach of the slide so adhesive is not required. But sections of fixed material

may detach of the slide, some adhesive is required.

Free Floating Sections

Sometimes frozen sections are picked on cover glass or these are directly smeared as free

floating sections. The idea of free floating sections is favoured by the fact that in certain

techniques like enzyme histochemistry, staining reagents are very costly and definitely free

floating sections save the staining reagents which is not possible when sections are taken on

slides or cover glass.

Merits of Cryostat

With the expansion of knowledge of histochemical studies and requirements of quick

reporting of emergency biopsies, cryostat has gained wide acceptance in most of the routine

laboratories. Cryostat is actually a Hospital device. Fresh emergency biopsies can easily and

promptly be sectioned in a cryostat. The results can be given by the pathologist while

surgery is still continuing and so it is possible for the surgeon to operate accordingly.