Impregnation

  

Impregnation (infiltration) is the process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will completely fill all the tissue cavities thereby giving a firm consistency to the specimen, and allowing easier handling and cutting of suitably thin sections without any damage or distortion to the tissue and its cellular components.

Embedding (casting or blocking) is the process by which the impregnated tissue is placed into a precisely arranged position in a mold containing a medium, which is then allowed to solidify.

The medium used to infiltrate the tissue is usually the same medium utilized for impregnation, and for general purposes is known as Embedding Medium.

There are generally three types of tissue impregnation, namely:

1. Paraffin Wax Impregnation

Paraffin is the simplest, most common and best embedding medium used for routine tissue processing.

Advantages:

a. Thin individual serial sections may be cut with ease from the majority of tissues without undue distortion.

b. The process is very rapid, allowing sections to be prepared within 24 hours.

c. Tissue blocks and unstained mounted sections may be stored in paraffin for an indefinite period of time after impregnation without considerable tissue destruction.

d. Many staining procedures are permitted with good results.

Disadvantages:

a.  Overheated paraffin makes the specimen brittle.

b. Prolonged impregnation will cause excessive tissue shrinkage and hardening, making the cutting of sections difficult.

c.  Inadequate impregnation will promote retention of the clearing agent. Tissues become soft and shrunken and tissue blocks crumble when sectioned and break up when floated out in a water bath.

d.  Tissues that are difficult to infiltrate, e.g. bones, teeth, brains and eyes, need long immersion for proper support; otherwise, they will crumble on sectioning. Prolonged immersion in paraffin, on the other hand, is not advisable.

e. Paraffin processing is not recommended for fatty tissues. The dehydrants and clearing agents used in the process dissolve and remove fat from the tissues.

After having been completely cleared, the tissue is submerged in two more changes of melted paraffin wax, either in a paraffin oven or an incubator, which has been regulated at 55 – 60^oC. Common waxes have melting points of 45^oC, 52^oC, 56^oC and 58^oC, the 56^oC wax normally used for routine work. In a laboratory with temperature ranging from 20 – 24^oC, paraffin wax with a melting point of 54 – 58^oC is indicated. If the laboratory temperature is between 15 – 18^oC, the melting point of wax to be used should be between 50 and 54^oC. Hard tissues require wax with higher melting point than soft tissues.

There are three ways by which Paraffin Wax Impregnation and Embedding of tissues may be performed:

a. By Manual Processing

At least four changes of wax are required at 15 minutes interval in order to ensure complete removal of the clearing agent from the tissue. The specimen is then immersed in another fresh solution of melted paraffin of approximately 3 hours to ensure complete embedding or casting of tissue.

The following is an example of time schedule for manual processing of tissues about 3 mm thick:

Fixation	10% Buffered Formalin	24 hours
Dehydration	70% Alcohol	6 hours
	95% Alcohol	12 hours
	100% Alcohol	2 hours
	100% Alcohol	1 hour
	100% Alcohol	1 hour
Clearing	Xylene or Toluene	1 hour
	Xylene or Toluene	1 hour
Impregnation	Paraffin Wax	15 minutes
	Paraffin Wax	15 minutes
	Paraffin Wax	15 minutes
	Paraffin Wax	15 minutes
Embedding	Paraffin Wax	3 hours

b. By Automatic Processing

This method makes use of an automatic tissue processing machine (i.e. Autotechnicon) which fixes, dehydrates, clears and infiltrates tissues, thereby, decreasing the time and labor needed during the processing of tissues, resulting in a more rapid diagnosis with less technicality. Usually, only 2 – 3 changes of wax are required to remove the clearing agent and properly impregnate the specimen. This is made possible due to constant tissue agitation that accelerates and improves tissue penetration giving rise to more consistent results.

One example of an automatic tissue–processing machine is the Elliot Bench–Type Processor.

The machine is mounted on rollers to permit the turning of platforms and easy access to beakers and wax baths. It makes use of 12 individual process steps, with ten 1–liter capacity glass beakers and two thermostatically controlled wax baths with a safety device cut–out switch to protect the wax against overheating. A transfer arm controlled by electric current moves the tissues from one processing reagent to another (by clock schedules). It can be removed by raising a spring–loaded plunger in the center of the cover plate, thereby allowing the tissue to be arranged manually anytime during the processing. A continuous vertical movement or rotation of the specimen carried accompanies agitation of fluid by a mechanism connected to the transfer arm. An electrical clock connected to a metal disc notched in positions of 15 minutes or more, serves to control the time needed for each processing step. The clock rotates and sets the transfer arm and mechanism into motion, moving the tissue to the next position. A delay mechanism is provided for, in instances where processing time may exceed 24 hours.

c. By Vacuum Embedding

Vacuum embedding involves the wax impregnation under negative atmospheric pressure inside an embedding oven to hasten removal of air bubbles and clearing agent from the tissue block thereby promoting a more rapid wax penetration of tissue. The time required for complete impregnation is thereby reduced from 25 – 75% of the normal time required for tissue processing. The tissue is not over–exposed to heat; brittleness, shrinkage and hardening of tissues consequent to overheating is therefore prevented.

This technique is particularly recommended for urgent biopsies, for dense and hard fibrous tissues (e.g., brain, connective tissues and bones), for lungs, spleen and other delicate tissues, e.g. eyes.

The vacuum embedding oven consists of a flat–bottomed heavy brass chamber covered with a heavy glass lid resting on a wide and thick rubber valve, which produces an airtight seal when the chamber is being used. The vacuum chamber is enclosed in thermostatically controlled water–jacket, usually maintained at a temperature of 2 – 4^oC above the melting point of the wax. There are two screw valves on the upper part of the chamber. One valve allows the readmission of air when the bath is under negative pressure. The other valve is connected to a tube, which in turn connected to a suction pump, to allow exhaustion of 400 – 500 mm inside, as shown in the manometer. A stopcock is provided to prevent water from being sucked back into the trap bottle and vacuum chamber when the water or suction pump is closed.

Factors affecting paraffin wax impregnation

Of the three methods of paraffin wax impregnation available, vacuum impregnation gives the fastest result. Total impregnation time, however, generally depends upon the nature and size of the tissue to be processed, and the type of clearing agents to be used. Larger and denser tissue blocks (e.g. bones, fibroids, brain) usually require longer periods and more frequent changes of wax. Benzene and xylene are easily removed from the tissues while chloroform and cedarwood oil are more difficult to remove and require more frequent wax changes. Addition of benzene may hasten displacement of cedarwood oil with less tissue shrinkage.

Precautions in Paraffin Wax Impregnation

Since prolonged treatment in melted paraffin causes shrinkage and hardening of tissues, making cutting difficult, the tissue should not be left in the medium for longer periods of time than is necessary. Infiltration in overheated paraffin (above 60^oC) will also produce shrinkage and hardening of tissues and destroy lymphoid tissues completely. To avoid this, the paraffin oven must be maintained at a temperature 2 to 5^oC above the melting point of paraffin to be used for impregnation.

Paraffin wax must be pure, i.e., free from dust, water droplets and other foreign matter. Fresh wax should be filtered before use in a wax oven at a temperature 2^oC higher than in melting point. Wax that has been trimmed away from the impregnated tissue may be melted and filtered for future uses, with a coarse filter paper, e.g., Green’s No. 904. When wax has been re–use, some amount of water inevitably is mixed with it; if excessive, this may impair the impregnating capacity of the medium and prevent formation of a good tissue block. Water must therefore be removed by heating the wax to 100 – 105^oC thereby raising its melting point. Paraffin wax may be used only twice, after which, fresh wax must be utilized.

When using an automatic tissue processing machine, wax usually becomes admixed with the clearing agent, especially in the first beaker, hence, water must be discarded.

For fixed knife microtome, a relatively hard wax with a higher melting point is recommended. Heavier microtome knives require harder paraffin wax than lighter ones.

Substitutes for Paraffin Wax

a. Paraplast

Paraplast is a mixture of highly purified paraffin and synthetic plastic polymers, with melting point of 56 – 57^oC. It is more elastic and resilient than paraffin wax thereby permitting large dense tissue blocks such as bones and brain to be cut easily with the same result as in double embedding. Blocks obtained are more uniform than with any other medium, with better ribboning of sections. Serial sections may be cut with ease, without cooling the tissue block, thereby preventing the formation of ice crystals artifacts. No deposit is left on the slide after staining, and no special processing schedule is required. It is soluble in common clearing agents and follows the same time schedule for paraffin impregnation, and does not tend to crack like other paraffin wax substitutes.

Embeddol is synthetic wax substitute similar to paraplast with a melting point of 56 – 58^oC. It is less brittle and less compressible than paraplast. Bioloid is a semi–synthetic wax recommended for embedding eyes. Tissue mat is a product of paraffin, containing rubber, with the same property as paraplast.

b. Ester wax

Ester wax has a lower melting point (46 – 48^oC) but is harder than paraffin. It is not soluble in water, but is soluble in 95% ethyl alcohol and other clearing agents; hence, it can be used for impregnation without prior clearing of the tissue. Cellosolve (ethylene glycol monoethyl ether) or xylene may be used as clearing agents, if indicated. In such instances, removal of the clearing agent must be gradual; that is, the tissue must be placed in a solution containing equal proportion of clearing agent and ester wax for 3 – 6 hours before finally transferring it to pure wax. Three to four changes of wax are required to ensure complete tissue impregnation. Sectioning of ester wax impregnated tissues should be done on a heavy duty microtome (e.g. sliding or sledge type microtome due to the relative hardness of the wax.

c. Water soluble waxes

These are mostly polyethylene glycols with melting point of 38 – 42^oC or 45 – 56^oC. The most commonly used is carbowax, a polyethylene glycol containing 18 or more carbon atoms, which appears solid at room temperature. It is soluble in and miscible with water, hence does not require dehydration and clearing of the tissue. The tissues are fixed, washed out and transferred directly into the melted carbowax. Processing time is reduced with the special advantage that harmful effects produced by dehydrating agents are consequently avoided. It does not remove neutral fats and lipids which are ordinarily soluble in reagents used for routine processing with paraffin, hence, allowing these substances to be demonstrated in thin sections. Tissues are not exposed to too much heat so that excessive hardening, shrinkage and brittleness of tissue is avoided; hence, making carbowax technique suitable for many enzyme histochemical studies. Cytologic details are excellently preserved.

For routine processing, four changes of carbowax, one each in 70% alcohol and 90% and 2 times in 100% concentration, at a temperature of 56^oC are used, at 30 minutes, 45 minutes and 1 hour (with agitation) respectively. Specimens are then embedded in fresh carbowax at 50^oC and rapidly cooled in a refrigerator.

Due to its hygroscopic nature, carbowax is very easily dissolved in water; hence care must be taken to avoid contact of the block with water or ice. Tissue sections are very difficult to float out and mount due to its extreme solubility in water, dehydrating and clearing agents. Adding soap to water or using 10% polyethylene glycol 900 in water will reduce tissue distortion and promote flattening and “floating out” of sections.

2. Celloidin Impregnation

Celloidin (colloidin) is a purified form of nitrocellulose soluble in many solvents, suitable for specimens with large hollow cavities which tend to collapse, for hard and dense tissues such as bones and teeth and for large tissue sections of the whole embryo. It is supplied in thin (2%), medium (4%) or thick (8%) solutions of cellulose dissolved in equal parts of ether and alcohol.

Advantages:

a. It permits cutting of tissue sections which are thicker than paraffin wax, and is therefore recommended for processing of neurological tissues.

b. Its rubbery consistency allows tissue blocks that are either very hard or of varying consistency, to be cut without undue distortion.

c. Dense tissues which are hard to infiltrate (e.g. bones and brains) and specimen which tend to collapse easily due to air spaces (e.g. eyes) are supported better, thereby avoiding the crumbling of tissues during sectioning. When eye sections are embedded by the paraffin method, the retina may be detached from the harder tissues (e.g. sclera and chancroid) that encircle it. The cedarwood oil used in the Dry Celloidin Technique helps to soften the brittle layers.

d. It does not require heat during processing; hence, producing minimum shrinkage and tissue distortion especially for cutting large bone sections. It is, therefore, recommended in cases when minimum shrinkage is required and frozen section technique cannot be done.

Disadvantage:

a. Celloidin impregnation is very slow (lasting for several days or weeks)

b. Very thin sections (less than 10u) are difficult to cut.

c. Serial sections are difficult to prepare.

d. Vapor of the other solvent is very inflammable; hence, it should never be used near an open flame.

e. Photomicrographs are difficult to obtain.

f.  It is very volatile and therefore must be kept in bottles with ground – glass stoppers to prevent evaporation.

There are two methods for celloidin impregnation of tissue:

a. Wet celloidin method – is recommended for bones, brain sections and whole organs.

After the usual fixation and dehydration of the tissue, it is place in equal parts of ether and alcohol for 12 – 24 hours. The tissue is then placed in thin celloidin (2 – 4%) for 5 – 7 days, transferred to medium celloidin (4 – 6 %) for another 5 – 7 days, drained off and poured with thick celloidin (8 – 12%) until the specimen has become impregnated, usually between 3 – 5 days. The specimen is removed from the celloidin, and transferred to an embedding medium containing freshly poured thick celloidin and kept in a tightly covered jar or dessicator in order to evaporate the alcohol – ether solvent. The dessicator top is removed for a few second, time and again, to admit fresh air and harden tissue block. Evaporation must be gradual to achieve a consistent, uniform degree of hardness throughout the block and prevent the formation of air bubbles.

When the ball of the finger leaves no mark on the surface of the tissue leaves no mark on the surface of the tissue block, evaporation and consequently, embedding, is considered to be complete. The tissue block is then stored in 70 – 80% alcohol until ready for cutting. This is done to avoid dehydration and shrinkage of tissues.

b. Dry celloidin method is preferred for processing of whole eye sections

The principle and procedure of this method is similar to wet celloidin method, except that 70% alcohol is not used for storage before cutting. Instead, Gilson’s mixture, made up of equal parts of chloroform and cedarwood oil is added to the celloidin block before hardening, to make the tissue transparent. The dry method does not make use of alcohol due to the presence of cedarwood oil in the block.

c. Nitrocellulose Method

Low viscosity nitrocellulose (LVN) is another form of celloidin soluble in equal concentration of ether and alcohol with a lower viscosity, allowing it to be used in higher concentrations and still penetrate tissues rapidly. Because of this, many workers prefer LVN to the ordinary celloidin for impregnation and embedding. It forms a harder tissue block and makes cutting of thinner sections possible. The tendency of tissues to crack may be prevented by adding plasticizers (e.g. Oleum Ricini or Castor oil) when embedding chrome–mordanted tissues.

Low viscosity nitrocellulose is more explosive than celloidin and should therefore be handled with care. When dry, striking or dropping the container will cause the substance to explode.  It is usually marketed while wet with alcohol. The container must be kept tightly covered and protected from sunlight to avoid evaporation of alcohol. When no longer needed for future use, the nitrocellulose should be carefully destroyed, since the material becomes increasingly dangerous as the alcohol continues to evaporate.

3. Gelatin Impregnation

Gelatin impregnation is rarely used except when dehydration is to be avoided and when tissues are to be subjected to histochemical and enzyme studies. It is used as an embedding medium for delicate specimens and frozen tissue sections because it prevents fragmentation of tough and friable tissues when frozen sections are cut. It is water–soluble, and does not require dehydration and clearing, although fixatives (such as 10% formalin) should still be washed out by running water whenever indicated. It has a lower melting point and does not cause overhardening of tissue by heating.

After the fixative has been completely washed out, the tissue is placed in 10% Gelatin with 1% Phenol for 24 hours, transferred to 20% Gelatin with 1% Phenol for the next 12 hours, and finally to another fresh solution of 20% Gelatin with 1% Phenol which is then allowed to cool in a refrigerator until impregnation and embedding are completed. Gelatin–embedded tissues are then transferred to 10% formalin for 12 – 24 hours in order to harden the tissue.

Tissues should not be more than 2 – 3 mm thick since gelatin–embedded specimens are harder to freeze than unimpregnated tissues. The 1% phenol serves to prevent the growth of molds.

Excess gelatin may be removed by floating the sections on to paper and trimming them with scissors. The volume of the impregnating medium should be at least 25 times the volume of the tissue.

EMBEDDING

After impregnation, the tissue is placed into a mold containing the embedding medium and this medium is allowed to solidify. Paraffin embedded tissues are arranged at the bottom of the mold together with their proper labels and immersed in melted paraffin at a temperature between 5 – 10^oC above its melting point and then cooled rapidly in a refrigerator at –5^oC or immersed in cold water to solidify. This allows hardening of tissues, giving them a firmer consistency and better support, thereby facilitating the cutting of sections.

The process by which a tissue is arranged in precise positions in the mold during embedding, on the microtome before cutting, and on the slide before staining, is known as orientation. Generally speaking, the surface of the section to be cut should be placed parallel to the bottom of the mold in which it is oriented.

Several types of blocking out molds may be used:

1. Leuckhart’s Embedding Mold – consists of two L–shaped strips of heavy brass or metal arranged in a flat metal plate and which can be moved to adjust the size of the mold to the size of the specimen. Blocks produced are even, with parallel sides, and with a fairly shaped initial setting of the wax. The mold is adjustable, to give a wide variety of sizes to fit the size of the tissue block for casting. It is recommended for routine use, although too slow and cumbersome for use in a busy laboratory.

2. Compound Embedding Unit is made up of series of interlocking plates resting on a flat metal base, forming several compartments. It has the advantage of embedding more specimens at a time, thereby reducing the time needed for blocking.

3. Plastic Embedding Rings and Base mold – consist of a special stainless steel base mold fitted with a plastic embedding ring which later serves as the block holder during cutting.

One model, the so called Tissue Tek, is a machine equipped with a warm plate to manage the impregnated specimen, and a cold plate at –5^oC for rapid solidification of the block. It consists of a white plastic cassette mold with detachable, perforated stainless steel hinge and snap–on lid, used to hold the tissue specimen throughout fixation, dehydration, clearing and wax impregnation.

The specimen is oriented on the base mold, the plastic embedding ring is placed in position and filled up with wax. Upon hardening, the tissue is taken out together with the embedding ring and is immediately ready for cutting without having to undergo trimming or mounting, thereby saving time and effort.

It produces easier orientation when resectioning of tissue is required. It is, however, very expensive and requires much greater space than the other embedding molds.

4. Disposable Embedding Molds

a. Peel–away disposable thin plastic embedding molds – available in 3 different sizes, are simply needed off one at a time, as soon as the wax has solidified, giving perfect even block without trimming. It may be placed directly in the chunk or blockholder of the microtome.

b. Plastic Ice Trays such as those used in ordinary refrigerators may be recommended for busy routine laboratories. Each compartment may be utilized for embedding one tissue block, which may then be removed by bending the plastic tray once the wax has solidified; or by smearing the inner mold with glycerin or liquid paraffin before embedding.

c. Paper boats are normally utilized for embedding celloidin blocks but are equally useful for paraffin wax blocks. They have the advantage of being cheap and easy to make. They provide easy and accurate identification of specimen, thereby avoiding confusion and interchange of tissue blocks. Rapid embedding of small or large volume of individual specimens is possible, since paper molds can be made to suit any size of tissue.

To mark the position of small tissues in the paraffin block, a mark such as an “X” is drawn with soft lead pencil on the inner surface of the bottom of the boat. This will attach and be visible on the wax block when solidified and removed from the paper boat.

Embedding molds should bear the case number and other identification data of the tissue block within. Once tissues have been embedded, they may be stored in a cool place indefinitely until they are cut.

Other embedding methods used are:

1. Celloidin or Nitrocellulose Method – recommended for embedding hard tissues such as bones and teeth and for large sections of whole organs like the eye, since the delicate layers of the eyeball are difficult to keep intact when other media are used.

Tissues may be embedded in shallow units of enamel pans which are covered by sheets of weighted glass. Bell jars could be used to control the rate of evaporation of the solvent.

2. Double Embedding Method – is the process in which tissues are first infiltrated with celloidin and subsequently embedded in paraffin mass. This is used to facilitate cutting of large blocks of dense firm tissues like the brain. They are also recommended for making small sections of celloidin blocks.

After being removed from the final dehydrating solution of absolute alcohol, the block is placed in equal parts of absolute alcohol and ether for 24 hours, then in celloidin 2% for 3 days after which it is drained of the excess celloidin and hardened and cleared in 2 changes of chloroform, each for 12 hours. It is then immersed in 4 changes of paraffin wax, for 1 – 2 hours each, finally embedded in fresh wax and cooled quickly.