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Frozen Section: Principle and Procedure

  • Pranab Dey
Chapter

Abstract

The frozen section is the rapid tissue section by cooling the tissue with the help of cryostat to provide immediate report of the tissue sample. The cryostat is the instrument to freeze the tissue and also to cut the frozen tissue for microscopic section. The rapid freezing of the tissue sample converts the water into ice. The firm ice within the tissue acts as embedding media to cut the tissue. The frozen section is mainly used for rapid diagnosis of the lesion for intraoperative management, to know the extent of the lesion, to do enzyme immunocytochemistry and immunofluorescence study and also to stain lipid and certain carbohydrate in the tissue. This chapter describes the principle, indications, techniques and various troubleshooting in frozen section. It also covers how to make good-quality section from frozen tissue section.

Keywords

Frozen section Cryostat Principle of frozen section Indications of frozen section Cryostat sectioning 

6.1 Introduction

The frozen section is the rapid tissue section by 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 the lesion or extent of the lesion at the time of operation. The cryostat is the instrument that has the arrangement to freeze the tissue and also to cut the frozen tissue for microscopic section.

6.2 Indications of Frozen Sections

The frozen section is used mainly for immediate diagnosis of the lesion for management and to know the extent of the lesion [1, 2, 3] (Box 6.1). It is also helpful to do enzyme immunochemistry and immunofluorescence study. At times, frozen section tissue is used for the demonstration of fat and carbohydrate in the tissue sample.

Box 6.1 Indications of Frozen Section

  • Rapid diagnosis of the lesion for intraoperative management

  • To know the extent of the lesion

  • To do enzyme immunocytochemistry

  • To do immunofluorescence study

  • To stain lipid and certain carbohydrate in the tissue

6.3 The Principle of Frozen Section

The rapid freezing of the tissue sample converts 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 firm, whereas increasing temperature makes the tissue softer.

Cryostat Machine Proper (Fig. 6.1)
Fig. 6.1

Cryostat machine with its parts

Temperature range in the machine: The cryostat machine has the usual temperature range from 0 °C to −35 °C. The most of the tissue is sectioned properly between −15 °C and −25 °C. The water-containing tissues can be sectioned in higher temperature, and fat-containing tissue needs much lower temperature to cut.

Rotary microtome (Fig. 6.2): Rotary microtome is placed inside the cabinet of the cryostat. Here the knife is fixed and the tissue is moved with the help of a rotary wheel.
Fig. 6.2

Microtome, blade, antiroll plate and tissue shelves are shown

Tissue shelf (Fig. 6.2): Just in one side of the microtome, there is a tissue shelf to keep the tissue. In this place the samples are kept for freezing. Usually the temperature of tissue shelf is lower than the overall cabinet temperature.

Place to keep the brush and knife holder: Just in front of the microtome machine, there remains a small place to keep the brush and knife holder.

Knife or blade: Nowadays, low- or high-profile disposable blades are used. The blade should be proper fixed to the holder to get an even pressure in the whole length. Alternatively Profile C steel blade is also used. The angle of the knife is kept in between 5° and 7°.

Antiroll plate (Fig. 6.2): Just in front of the knife, there is an antiroll plate that prevents the rolling of the cut tissue. It is usually a glass plate within a metal frame. The undersurface of the plate has free space, and there is a gap between the knife and the plate.

Alternating to antiroll plate, a cool sable hair brush can be used to get unrolled tissue.

Specimen holder: The specimen holder or chuck is supplied by the manufacturers in different sizes and shapes. Usually these are round metal structures.

Embedding medium: This medium is used to hold the tissue over the chuck. Presently optimum cutting temperature (OCT) compound is used as embedding medium. The OCT is made of water-soluble glycols and resin.

6.4 Cryostat Sectioning

The process of the cryostat sectioning needs the following steps.
  1. 1.
    Grossing and cutting the specimen (Box 6.2): The cutting surface of the tissue should be smooth. The following steps in grossing of the tissue are mandatory for accurate reporting:
    • Identify the tissue sample of the patient and the requisition form: This is the first and foremost part of the frozen tissue grossing.

    • Salient clinical information: The essential clinical information is very helpful as it guides the pathologist to reach the possible differential diagnosis.

    • Tissue appearance: The gross appearance of the tissue such as colour, texture, consistency and any suture to mark the anatomical position.

    • Resection margin: It is very important to identify the resection margins of the tumour. The resection planes and margins should be inked thoroughly. The different colours of ink can be used for medial and lateral margin identification.

     

Cutting the tissue: The tissue should be fresh without any fixative. The tissue should be preferably dry, and it should not be wrapped in gauze piece. Any suture, staple or sharp hard structure should be removed from the tissue sample. Now the tissue is cut into small pieces as it facilitates freezing. Take multiple sections of the tissue to understand the main pathology and to minimize the error. Use a new sharp scalpel blade, and first cut the most important area that needs microscopic examination. It is preferable to use gentle stroke of the scalpel rather than too much pressure.

Cytology of the tissue: At times the imprint of the tissue on the slide provides good morphological details such as lymphoma of the lymph node. Similarly crushing of tissue also provides excellent morphological details such as in case of tissue of the brain tumour.

Box 6.2 Grossing for Frozen Section Tissue

  • Identify the tissue sample of the patient.

  • Clinical information: provides possible differential diagnosis.

  • Tissue appearance: colour, texture, nodule, any suture.

  • Anatomy of the tissue: identify the resection planes and margins.

  • Colour the resection planes and margins.

  • Section cutting:
    • Use sharp blade.

    • First cut the most important area.

    • Give gentle pressure and avoid too much pressure.

  • Cytology preparation: if needed make
    • Imprint smear

    • Scrape smear

    • Crushed smear

  1. 1.

    Tissue embedding in the mould (Fig. 6.3): The small piece of the tissue is kept in the centre of the mould, and then the OCT is poured over it in excess. Then the tissue holder or chuck is firmly placed over the tissue with overflown OCT.

     
  2. 2.

    Tissue loading in the frozen section chamber: The tissue is now placed in the frozen section chamber, and cold spray can be used to make the process faster.

     
  3. 3.

    Loading the blade: The cutting knife or blade is now loaded and the proper alignment is done.

     
  4. 4.

    Trimming the tissue: The loss of normal or natural colour to whitish colour indicates that the tissue is frozen. The frozen tissue in the tissue holder is now placed in the holder of the microtome. The block is trimmed to remove the excess OCT and to get the smooth tissue surface for sectioning.

     
  5. 5.

    Sectioning (Fig. 6.3): The tissue is now cut gently and is spread over the antiroll plate with the help of a brush. The brush should be cooled. The tip of the tissue is guided by the brush.

     
  6. 6.

    Section lifting: The glass slide of normal room temperature is pressed firmly over the tissue section, and normally the tissue sticks immediately.

     
  7. 7.

    Fixation: The tissue should be immediately fixed in methanol for 1 min or 95% ethanol for few seconds. Rapid fixation within few seconds is mandatory. In case of delayed fixation, the cells are swollen, and the cytoplasmic margin may be ruptured giving hazy appearance of the margin of the cells.

     
Fig. 6.3

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

Troubleshooting in frozen section: Various problems may arise during the cutting of frozen section tissue. This has been highlighted in Table 6.1.
Table 6.1

Troubleshooting in frozen section

Problems

Cause

Solution

Freezing artefact

Formation of ice crystal within the tissue. Water-containing tissue shows more such artefact

• Freeze the tissue rapidly, i.e. snap freezing

• The tissue specimen should not be in saline solution before freezing

Uneven tissue embedding

The surface of the tissue is uneven, and the vital information may be lost

• Make tissue even at the cutting surface before freezing

Block is loosen during chucking

The chuck may be too cold when the tissue is placed on it

• Take the tissue out and reattach it on a clean chuck which is not too cold

Tissue crumpled

The tissue in the block is warm or too cold

• Make the block of tissue in the optimum temperature: −15 °C to −20 °C

Chattering artefact

The temperature of the block is too cold, and the tissue becomes hard. The blade will cut the tissue thick and thin in regular interval

• Bring the block in optimum temperature. Pressing the cut surface of the block by gloved finger may make the block warmer

Thin stripe in tissue

The perpendicular tear in the tissue is due to nicks on the blade

• Replace the blade by a sharper one

Widely striped tissue and also tearing of the tissue

This may happen if the tissue is sticking with the blade

• Clean the blade or replace with a new one

Fixation: Immediate dip in 95% ethyl alcohol for a few seconds fixes the tissue.

6.5 Staining

We commonly use haematoxylin and eosin (H&E) and toluidine blue stain.

6.5.1 H&E Staining

  • Rinse the slide in tap water.

  • Put in haematoxylin for 1 min.

  • Rinse in tap water for 5 s.

  • Rinse in Scott’s tap water for 5 s for bluing.

  • Dip in eosin for 20 s.

  • Rapidly rinse in tap water.

  • 95% ethanol for 10 s.

  • 100% ethanol for 10 s.

  • 100% ethanol for 10 s.

  • Dip in xylene for 20 s.

  • Mount by DPX.

6.5.2 Toluidine Blue Stain

This is a very simple stain and takes only a few seconds. The drops of toluidine blue stain are put on the section, and the coverslip is put on the section. The slide is now ready to see. The histopathologist feels more comfortable in H&E stain than this unfamiliar toluidine blue stain.

6.6 Factors Affecting the Good-Quality Section

The common factors responsible for the good-quality smear include:
  • Temperature: when the temperature falls, water within the tissue becomes frozen and gives the tissue hard consistency. The optimal temperature of frozen tissue is in between −15 °C and −25 °C. Warm tissue remains soft and sections crumple. On the other hand, the overcooled tissue becomes very hard and brittle and produces again bad-quality crumpled section. Moreover the hard tissue may cause “chattering” artefact and also thick and thin sections. Different tissue contains variable amount of fat and water. The consistency of different tissue varies, and therefore the optimum temperature to cool the tissue varies considerably. Table 6.2 shows the optimum temperature of different organs to have good frozen section.

  • Tissue consistency: other than the optimum cooling temperature, the consistency of tissue has significant effect on cutting such as:
    1. (i)

      Fatty tissue: It is difficult to cut the fatty tissue in frozen section. Fat may smear on the knife and may make problem in cutting.

       
    2. (ii)

      Collagenous tissue: The firm collagenous tissue is difficult to cut.

       
    3. (iii)

      Necrotic tissue: Soft necrotic tissue may create considerable problem as they may fall from the slide making hole in the section. It is preferable to take only viable tissue for frozen section.

       
    4. (iv)

      Bony hard tissue: Hard tissue like bone or cartilage may damage the blade significantly. In this situation a new section can be processed, or new blade can be used.

       
  • Tissue size: The size of the tissue sample should be small as the larger tissue takes much longer time to freeze.

Table 6.2

Optimum temperature for frozen section

Tissue

Optimum temperature

Brain, liver, spleen

−7 °C to −10 °C

Rectum, uterus, adrenal, muscle, skin

−12 °C to −15 °C

Heart, lung, intestine, pancreas, ovary, cervix, prostate

−16 °C to −20 °C

Bone marrow, breast

−20 °C to −25 °C

References

  1. 1.
    Ayhan A, Ozler A, Dursun P, Haberal AN. Potential role of increasing number of sections in frozen section diagnosis of ovarian tumors. J Exp Ther Oncol. 2016;11(4):245–50.PubMedGoogle Scholar
  2. 2.
    Chambers KJ, Kraft S, Emerick K. Evaluation of frozen section margins in high-risk cutaneous squamous cell carcinomas of the head and neck. Laryngoscope. 2015;125(3):636–9.CrossRefPubMedGoogle Scholar
  3. 3.
    Hatami H, Mohsenifar Z, Alavi SN. The diagnostic accuracy of frozen section compared to permanent section: a single center study in Iran. Iran J Pathol. 2015;10(4):295–9.PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • Pranab Dey
    • 1
  1. 1.Education and Research (PGIMER)Post Graduate Institute of Medical Education and Research (PGIMER)ChandigarhIndia

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