Lab 3: Section Cutting and

Frozen Section

Objectives :

1. To ensure good section cutting and frozen section.

2. To overcome troubleshooters during section

cutting and frozen section.

3. To familiarize the staff with the equipment used

for section cutting and frozen section.

Overview:

Tissues are sectioned using a microtome.

Turn on the water bath and check that the temp is 35-37ºC.

Use fresh deionized water (DEPC treated water must be used if in situ hybridization will be performed on the sections).

Blocks to be sectioned.

Place a fresh blade on the microtome; blades may be used

to section up to 10 blocks, but replace if sectioning becomes

problematic.

Insert the block into the microtome chuck so the wax block

faces the blade and is aligned in the vertical plane.

Cont.

Face the block by cutting it down to the desired tissue plane and

discard the paraffin ribbon.

If the block is ribboning well then cut another four sections and pick

them up with forceps or a fine paint brush and float them on the surface

of the 37ºC water bath.

Float the sections onto the surface of clean glass slides.

If the block is not ribboning well then place it back on the ice block to

cool off firm up the wax.

If the specimens fragment when placed on the water bath then it may

be too hot.

Cont…

Place the slides with paraffin sections on the

warming block in a 65°C oven for 20 minutes

(so the wax just starts to melt) to bond the tissue

to the glass.

Slides can be stored overnight at room

temperature.

Devices for cutting sections

Microtome

VibratomeCryostat

Cont…

Most sectioning in routine histopathology

departments is done with a microtome producing

sections of ~3μm thickness, from tissue that has been

embedded in wax.

A number of devices are available for cutting

sections.

Microtomes

These are mechanical devices for cutting uniform

sections of tissue of appropriate thickness.

Ultramicrotome >>>> 50-100nm

Types of microtome

1. Hand microtomes

2. Rocking microtome.

3. Rotary microtome.

4. Freezing microtome.

Cont.

5. Base sledge microtome.

6. Vibrating knife microtome.

7. Sliding microtome.

Rotary microtome

Designed for cutting celloidin-embedded tissue

blocks.

The Knife or blade is stationary, specimen slides

under it during sectioning.

Also used for paraffin-wax embedded sections.

Section adhesives

An adhesive is a substance which can be smeared on

to the slides so that the sections stick well to the slides.

Most of the tissue sections which are adequately thin

and thoroughly dried without any air bubble trapped

under them do not require an adhesive, as in case of

routine H and E staining.

Cont…

But for histochemical methods requiring alkaline solutions, e.g.

ammonia tend to remove sections from slide for such cases

adhesive is required.

Also adhesive is required for tissues like brain, spinal cord,

blood clot, decalcified tissues which have a tendency to detach

themselves from the slide.

Tissue impregnated with ester wax also requires section

adhesive.

Types of adhesive

Albumin

Gelatin

Starch

Cellulose

Resin

Poly L Lysine

Common Problems with Sectioning Cutting Problems:

1. Cut on an angle:

Angled cuts can be identified in the following ways: The section or cells within it are oval in shape.

One can focus through several cell layers in one

area of the section.

Part of the section appears to be “smeared”.

Cont…

2. Cut too thin: This problem can be identified if a section

has a part missing, and/or it is not completely round.

3. Cut too thick: This problem can be identified if

boundaries within the section appear exceptionally thick, or

if you are able to focus through several cell layers across the

whole section.

Cont…

4. Sections cut in half: The problem here is that there are

too many sections in the drop of water, and convection is

bringing them back towards the razor where they are

being cut in half.

Frozen Section

A thin slice of tissue that is cut from a frozen specimen and

is often used for rapid microscopic diagnosis section and a

histologic section of tissue that has been frozen by exposure

to dry ice.

The frozen section procedure is a pathological laboratory

procedure to perform rapid microscopic analysis of a

specimen. It is used most often in on cological surgery.

Cont…

The technical name for this procedure is cryosection.

The quality of the slides produced by frozen section is

of lower quality than formalin fixed, wax embedded

tissue processing.

While diagnosis can be rendered in many cases, fixed

tissue processing is preferred in many conditions for

more accurate diagnosis.

Uses from frozen section:

The principal use of the frozen section procedure is the

examination of tissue while surgery is taking place. This

may be for various reasons:

In the performance of Mohs surgery - a simple method for 100% margin control of a surgical specimen.

Mohs Surgery Mohs surgery, also known as chemosurgery, developed in 1938 by a general surgeon, Frederic E. Mohs, is microscopically controlled surgery used to treat common types of skin cancer. During the surgery, after each removal of tissue and while the patient waits, the tissue is examined for cancer cells. That examination informs the decision for additional tissue removal. 

Cont. If a tumor appears to have metastasized, a

sample of the suspected metastasis is sent for cryo section to confirm its identity.

If the tumor has metastasized, surgery is usually not curative, and the surgeon will choose a more conservative surgery, or no resection at all.

Cont…

If a tumor has been resected but it is unclear whether the

surgical margin is free of tumor, an intraoperative

consultation is requested to assess the need to make a

further resection for clear margins.

In a sentinel node procedure, a sentinel node containing

tumor tissue prompts a further lymph node dissection,

while a benign node will avoid such a procedure.

Cont.

If surgery is explorative, rapid examination of a lesion

might help identify the possible cause of a patient's

symptoms.

Rarely, cryo sections are used to detect the presence of

substances lost in the traditional histology technique, for

example lipids. They can also be used to detect some

antigens masked by formalin.

Embedding the tissue

The selected piece of tissue is then placed on a metallic holder

and must be oriented a certain way so that the future section will

reveal proper spatial relationships, this orientation depends on

the question being asked.

Sometimes orientation is not important; at other times it is of

paramount importance.

The tissue is embedded in OCT mounting medium and is then

placed either in cooled 2-methyl butane or the cryostat machine

where it is properly frozen.

Cryostat: The machine, which cuts the tissue, is the

cryostat. Certain things should be routinely checked in the

operation of this machine:

a) Temperature: The temperature should be at -20°F for

most tissues.

For tissues with a large fat component, -40°F is optimal.

This temperature is critical for optimal sectioning.

Cont…

Too high, i.e., -10°F and the tissue will not

stay frozen and firm and will not cut crisp.

Too cold, i.e., -50°F and the tissue will

crumble and become powder. The Ideal tissue

should cut like butter, smooth and in one piece.

b) Blade sharpness and angle: The blade should be sharp and should

be changed approximately once every 2 weeks.

A dull blade cuts dull.

Equally important is the blade angle. There is an optimal angle between

blade and tissue:

Too steep an angle and the tissue will crumble like it was too cold.

Too shallow, then two things will happen. The section will

alternately skip and not cut and then it will cut, but too thick.

Methodology

GOOD LUCK