Section Cutting in histopathology

Section Cutting in histopathology

MICROTOMY

Having fixed, processed and embedded the tissue, the next stage is to cut sections from the block. The machines or instruments used to cut thin sections are called microtomes.

MICROTOMES

The earliest recorded form of microtomes was a non-mechanical, hand operated device which was nothing more than an elaborated scalpel. The first truly mechanical 

microtome was perhaps the sliding microtome devised by Adams in 1798. Microtomes are classified into various types, but all of them should fulfil the following requirements:

(a) Rigid support for the knife and the tissue block. 

(b) Means of moving either the tissue block across the fixed knife-edge or the knife edge across the block. 

(c) Means of accurately advancing the tissues to cut each section at the predetermined thickness. 

The choice of microtome depends on the type of work, the nature of the tissue preparation and embedding.

 FOR KNOW MORE  KNOWLEDGE ABOUT  MICROTOMES AND THEIR TYPES - CLICK HERE

MICROTOMES 

1. Sliding microtome

2. Base sledge microtome

3. Cambridge rocking microtome

4. Rotary microtome

5. Freezing microtome

6. Cryostat

Ultra-microtom

MICROTOME KNIVES

(a) Planoconcave profile

(b) Wedge-Shaped

(c) Biconcave Profile

(d) Tool-Edge Profile

TERMSUSED IN MICROTOMYY

Tilt of the microtome knife

Compression

Rate of cutting

Ambient temperature

Orientation

Slant of the knife

• SHARPENING OF MICROTOME KNIVES

Hand Sharpening

1. Belgian yellow stone

2. The Belgian black vein (blue-green)

3. Arkansas

4. Aloxite

Procedure of Honing

Plate glass honing

Knife Sharpening Machines (Automatic Hones)

Plastic blades

Factory grinding

STROPPING

 

ROUTINE PARAFFIN SECTION CUTTING

The block having been attached to the wooden holder, is now fixed in the microtome. The knife is inserted and secured firmly with tightening screws. The block is moved forward until the tissue surface is almost touching the knife edge. The surface of the tissue block is pared off in 15-25 um slices to expose the tissue face before taking sections. This rough trimming is carried out either using one end of the knife or with an old microtome knife that is specifically kept for that purpose. Keep the block in ice while resetting the knife.

Set the microtome gauge for the desired thickness, which routinely is 4-6um. Remove the block from the ice, wipe dry and fix firmly in the clamp or chuck of the microtome. Cutting is carried out with regular even strokes. The general rule is that hard tissue, e.g., thyroid, is best cut with firm, quick strokes, while soft tissues will require slow gentle strokes. Lymph nodes and nervous tissues should be cut slowly with even regular strokes. It should be noted that the harder the tissue, the cooler the block should be, so that the resistance between the knife and the wax and the tissue will be as even as possible. Ideally, the disparity between the consistency of the tissue and that of the wax should be minimal. The microtome is operated until complete sections are being cut and a cutting rhythm is being maintained.

Using a diamond pencil, glass slides are numbered, the numbers corresponding to those on the blocks to be cut. To minimise mistakes and confusion, frosted end slides are used and numbered with pencil as each block is cut. The slides are wiped clean with fine soft cloth and the numbered surface of each slide is smeared with a thin coat of adhesive mixture which is spread with the pad of the finger.

Ribbon Sectioning

To obtain ribbons of sections, it is important that the upper and lower surfaces of the block are parallel. Cutting of ribbon sections is an eloquent testimony of experience of a good microtomist. The first section is raised carefully with the index finger or flat tipped forceps or a pencil or camel's hair brush. As the knife makes contact with the block to start cutting the next section, locally generated pressure welds the edge of the second section to the edge of the first section. This continues with succeeding sections with the result that a ribbon of sections is formed. An experienced section cutter can produce a ribbon of up to 10 inches long. The sections can be laid out on a cardboard sheet in a serial order. The ribbons are then cut into suitable lengths.

 Usually, the sections do adhere to the cardboard at the time of cutting and they are released by gently passing the scalpel blade obliquely under the line of adhesion. The cut sections are floated out in the water bath. It is also the usual practice to float out a ribbon of about 4-6 sections and with the aid of two dissecting needles, unwanted sections are flicked out. The sections may be destroyed if the needle tips are not clean and care must be taken in picking up the sections.

Adhesive Mixture for Coating Slides

The adhesive mixtures are all protein solutions. It is believed that these agents act mostly by reducing the surface tension thereby producing closer capillary adhesion of the sections to the slides.

For routine staining procedures, these adhesives are not very necessary as long as the slides are clean and grease-free. However, they are needed where the methods involve great exposure to actions of acids and strong alkalis (, e.g., ammoniacal silver solutions), when dealing with complex staining methods and also for such tissues as bone and nervous system. Being protein, the amount used to coat slides must be very small in order to prevent the tissues being retained by the protein. The addition of thymol as preservative in the mixture prevents bacterial contamination which might present confusion in Gram and PAS stains.

Mayer's egg albumin glycerol This is the most popular adhesive mixture. It is composed of 50 ml of white of fresh egg and 50 ml of glycerol which are mixed and then filtered through several layers of gauze. Thymol (100 mg) is added as the preservative. Some workers prefer to dilute the Mayer's solution with 50 ml of sterile distilled water.

3 Aminopropyltriethoxysilane (APES)

This is the best section adhesive. Clean slides are dipped in 2% APES in acetone and drained two times and finally dipped in distilled water. The slides are stored for long time and used when needed.

FLOATING OUT BATH

Ideally, the bath should be close to the microtome. The circular, thermostatically controlled bath, 10 to 12 inches in diameter and 3-4 inches in depth, is widely used. The inside surface is black and this enables the section to be easily seen in the bath.

The temperature of the bath should be set at 45°C, usually 10°C below the melting point of wax used for blocking out. The bath should be filled with water to within 1/2-1 cm from the top. At the end of the day, the bath should be disconnected, emptied and thoroughly wiped clean. If a scum appears on the surface of the water before or during cutting, it may be removed by gently placing a large sheet of filter paper or any other suitable absorbent paper on the surface of the water and then pulling the paper off, with the edge held in the fingers just a little above the water surface.

Floating out Techniques

1. Place the section on a slide.

2. Place one or 2 drops of 20% alcohol beside the section. The alcohol flows underneath the section making the section to float on the fluid.

3. With fine needles, carefully tease out the section if there are folds.

4. Gently lower the section onto the bath. Floating out helps remove folds in sections as the heat affects the wax, thus flattening the section.

5. With the labelled coated slide, pick up the section from the bath: Immerse the slide almost vertically in the water bath. The coated surface of the slide is moved very close to the section. The slide is then raised gently in an even motion as it touches the edge of the section, the section attaches itself to it as the slide is drawn upward.

6. Place the slide on hot plate (45°C) to dry before staining.

DIFFICULTIES ENCOUNTERED IN PARAFFIN SECTION CUTTING

Some faults are invariably encountered in the cutting of paraffin sections. Difficulties are usually due to one of the following causes (Table 5.1 ). Difficulties due to improper fixation Poor fixation results in the production of soft, mushy tissue block and sections that crumble when cut. This is most common in very mucinous tissues, e.g., Pseudomucinous adenomas of the ovary where the fixative fails to penetrate the viscous mucus. To overcome this difficulty, the tissue should be properly fixed in buffered neutral formalin, using a thin block, prior to washing with normal saline to remove excess of mucus. On the other hand, the production of hard brittle blocks is due to prolonged fixation in fixatives such as Zenker's or Helly's fluid, and it may lead to the production of frag. mented sections. Such blocks often cause the knife to 'ring' or 'chatter' i.e. vibrate. This may lead to the production in the section of lines or scores that run parallel to the knife edge, or alternate thick and thin sections. Very hard blocks can be softened by soaking overnight in molifex.

 Difficulties due to faulty dehydration, clearing and embedding The clearing agent turns milky when a poorly or insufficiently dehydrated tissue is placed in it. This results in the incompletion of the processing; and blocks of such tissues are usually soft and mushy. It is difficult to cut sections from such blocks as the sections tend to fray and crumble. If insufficient dehydration is noticed during clearing, it can be corrected by returning the tissue for further treatment in absolute alcohol.

Inadequate clearing makes the tissue opaque or cloudy and so makes sectioning almost impossible. This fault can be remedied by returning the block to the wax bath until the wax is completely melted and then returned to the clearing agent for further clearing. It is sometimes easier and faster to select another block of tissue and process. On the other hand, prolonged exposure to certain clearing agents, e.g., xylene, makes the tissue excessively hard and brittle and may also lead to shrinkage. Poor impregnation with wax produces a "moist block that crumbles during sectioning. At the other end, prolonged wax impregnation will result in very hard and shrunken tissue block. 

Difficulties due to the tissue Following routine processing, certain tissues, e.g., blood clot. cervix and thyroid, tend to become very hard. Such

tissues should be cooled with ice before cutting. The cutting should be done with firm short strokes. Clearing such tissues in chloroform minimises hardness.

Subcutaneous tissues, breast lipomas and other fatty tissues tend to give soft blocks and shredded sections. This is due mainly to inadequate removal of fat. These kinds of tissues are better impregnated in the vacuum bath and the blocks selected should not be more than 2 mm thick. Hardness and brittleness are also conferred on brain and lymph nodes when clearing is done with xylene. To prevent this difficulty, chloroform is best employed for clearing and impregnation carried out in the vacuum bath.

Soft tissues experience about 15% shrinkage as a result of their being compressed by the setting and cooling of wax block. Sections of such tissues are compressed further when sections are cut. When being floated out, the sections tend to expand more than the sections of firmer tissues. To prevent creases and wrinkles which may occur, the outer rim of wax should be split with a dissecting needle.

Difficulties due to the microtome knife The knife when not properly clamped in the microtome, will tend to jump, especially on striking hard tissues. Also, excessive tilt of the knife (i.e. too great a clearance angle) will make it vibrate and 'chattering' is likely to occur. This creates lines of unequal thickness parallel to the knife edge in the sections. Again, if the tilt of the knife is very great, scraping instead of cutting will occur and it may be impossible to obtain a section. In addition to correct setting and adequate clamping, the heavier the knife, the less tendency to vibration.

Sections of unequal or irregular thickness may result from inadequate clearance angle (which leads to the compression of the block) or from inadequate tightening of the block holder.

The knife should be sharp, as a blunt knife never produces good sections. When the width of the section is much less than that of the block, the knife has probably lost its bevel. If there are serrations

or minute nicks in, or wax adhering to the back of the knife edge, it will score or scratch the sections in the direction of the cutting stroke. Similar scratches may be due to particles of dust in the wax or by minute spicules of calcium or silica in the tissue. 'Ribboning is difficult to produce with a really blunt knife.

Many other faults in cutting sections may be due to the wax or the thickness setting of the microtome. Too hard a wax makes ribboning difficult; and folds and wrinkles are very likely to occur in the case of softer tissues. If the sections are cut too thick (more than 8 um), they tend to roll up or fracture, whereas very thin sections (less than 4 um) require gentle even strokes during cutting. When the knife edge or wax block is too warm, the section lifts from the knife on the upstroke (rotary microtome) or backstroke (sliding microtome with fixed knife) This prevents ribbon formation.

FROZEN SECTIONS

Preparation of Sections

1. Place a drop of water in the centre of a pre cooled block holder.

2. Place the tissue to be sectioned in the drop of water.

3. Rapidly freeze the tissue--this is done by either standing the block holder in a bath of alcohol or acetone containing dry ice or by placing the block holder in the special freezing attachment and exposing the tissue to carbon dioxide gas.

 4. When the tissue is frozen, position the holder in the microtome.

Note

1. The block-holders are stored in the cryostat to be used when required.

2. OCT (optimal cutting temperature) compound can be used to cover the block-holder and placed in fastfreeze shelf of the cryostat to effect fast freezing of the tissue.

Cutting of Section

1. Adjust the tissue correctly to the microtome.

2. Trim the tissue with the aid of the remote control outside the chamber.

3. About 15-30 minutes before cutting starts, place the knife in position in order to attain the correct cutting temperature.

4. Set the section thickness control (usually at 5-10 um ) and the automatic advance mechanism.

5. Position the anti-roll plate so that its edge is parallel and almost even with the knife edge

(about 70 um gap between them).

6. Allow the temperature in the chamber to equilibrate by closing the cabinet for about

2-3 minutes.

7. Make sure the slides and stains are ready.

8. Cut section with a slow steady motion, but some harder tissues are best cut with firm fast strokes.

9. With skilled technique, the sections will move smoothly underneath the anti-roll plate.

Mounting Frozen Sections

The sections having been cut the cabinet is opened and the anti-roll plate is flipped back. A clean slide (or cover glass) is then carefully lowered onto the section which gets attached to the slides. This attachment of section to slide can be facilitated by the use of special holder fitted with a section cup. For sections of fresh unfixed tissue, no adhesives are required- just a few seconds of waving in the air is sufficient. Sometimes, it is necessary to fix the section and this is done as soon as the section is cut. The best fixing solution is formal-alcohol (15 ml of 40% formaldehyde in 85 ml of 95% ethanol, optionally 0.5 ml glacial acetic acid may be added). Usually, the fixed sections may detach from the slide during staining. For this reason, they are best picked up with slides coated with albumen or any other suitable adhesive.

FIXING TISSUE FOR CRYOSTAT EXAMINATION

The main purpose of the cryostat is for rapid surgical diagnosis and so time is saved using unfixed, usually fresh, tissues. Sections from such tissues adhere well to clean slides without adhesives. Also, enzymes are well preserved in the unfixed frozen sections.

It is however possible to use fixed tissue in cryostat examination. The tissue block selected by the pathologist during cut up, may be put in boiling buffered formalin for about 2-3 minutes and cooled before freezing and sectioning for rapid diagnosis. Shrinkage is a common problem and the firmness of the block is usually insufficient. A suitable fixative for this technique is 10% formal calcium at 4°C. It is important to wash tissues fixed or stored in alcohol in water for about 18-24 hours since alcohol inhibits freezing. It is equally important to remove mercury chloride pigment and potassium dichromate by the usual methods before being subjected to the cryostat.

Stains for cryostat sections Though there are many staining methods employed for frozen sections, two methods are very popular. These are the

haematoxylin--eosin and polychrome methylene blue methods.

 Haematoxylin and eosin method Sections are air-dried for 1/2-1 minute. For convenience, the staining solutions are arranged in sequence in a series of coplin jars on a tray.

1. Fix the air-dried section in pure acetone for 20 seconds.

2. Place the section in absolute alcohol for 1/2 minute to 1 minute.

3. Place in 95% ethanol for

5 seconds.

4. Place in distilled water until no longer 'greasy' or cloudy.

5. Place in Harris's haematoxylin for 1-2 minutes.

6. Place in distilled water with agitation for 5-10 seconds.

7. Dip in 0.5% sodium borate until blue.

8. Place in 70% ethanol for 5 seconds.

9. Place in 1% alcoholic eosin for 5-20 seconds.

10. Wash in water 10-30 seconds.

11. Dehydrate through graded alcohols.

12. Clear in 3 changes of xylene and mount in DPX, Clarite, or HSR.

Theory of staining next page