Embedding in Histopathology: Embedding is the process in which the tissues or the specimens are enclosed in a mass of the embedding medium using a mould.
Since the tissue blocks are very thin in thickness they need a supporting medium in which the tissue blocks are embedded. This supporting medium is called embedding medium. Various embedding substances are paraffin wax, celloidin, synthetic resins, gelatine, etc.
Embedding in Histopathology
The choice of embedding media depends upon
- Type of microscope
- Type of microtome
- Type of tissue eg. hard tissue like bone or soft tissue like liver biopsy
Paraffin wax with a higher melting point (56 to 62oC) is used for embedding. The molten wax is filtered inside the oven through a course filter paper into another container. This will protect the knife edge.
Other Types of Embedding Media
Carbowax: It is a water soluble wax. Therefore tissues are directly transferred to water soluble wax after fixation and washing.
Methacrylate: It is easily miscible with alcohol and gives a clear and hard block when polymerised Polymerization takes place in the presence of a catalyst. Any trace of water causes uneven polymerization and formation of bubbles in the block around the tissue.
Epoxy Resin (Araldite): Epoxy polymers of araldite is used in higher resolution work and to see greater details. Epoxy resins are used for electron microscopy. Epoxy polymers of araldite differ from methcrylate in that they are crosslinked causing the cured solid block of araldite to be insoluble in any solvent. Longer filtration is required because the viscosity of resin is greater than methacrylate.
For electron microscopy araldite is obtained as casting resinCY212, ahardner DDSA and an amine accelerator, DMP (ditrimethylamino methyl phenol) Blocks are suitably cured before sectioning for 48 to 60 hours at 60oC.
Agar embedding: It is mainly used in double embedding. Multiple fragments and friable tissue may be impregnated in one block when sectioning on the cryostat. Another use of agar embedding is for FNAC specimens. ‘Embedding in Histopathology’
Celloidin media: Celloidin is a purified form of nitrocellulose. It is used for cutting hard tissues.
Gelatin: Its melting point is less than the melting point of agar. Gelatin may be used when frozen sections are required on friable and necrotic tissues. ‘Embedding in Histopathology’
TYPES OF MOULDS
A variety of moulds are used for embedding. These may be LEUCKHARD embedding moulds (L mould) paper blocks, plastic moulds. Most of the laboratories use L moulds (Fig-1). L moulds are made up of metal, easy to procure, reusable and may be adjusted to make different size of blocks. One limb of the”L” is longer than the other. The two “Ls” are jointed to form a sides of the rectangular box that act as a cast to make the mould.
Plastic moulds: Most of the laboratories use plastic embedding rings now. These are relatively inexpensive, convenient and support the block during sectioning and are designed to fit it on the microtome. This eliminates the step of mounting or attaching the block on a holder (metal or wooden holder).
- Tissue-Tek System1 or Mark1 system: In this system plastic embedding rings (Fig-2) with stainless steel moulds (Fig-3) allow rapid embedding and cutting of tissues. In this system the blocks are stored with the plastic rings; the angle does not change for further requirement of sections. The disadvantage of this method is that the space required for storing is more. ‘Embedding in Histopathology’
- Tissue-Tek system 2 or Mark 2 system: The Mark 2 system has provided a cassette to hold tissue during processing and has a stainless steel lid on the plastic cassette. The cassette has a rough surface on one side of it with a slope where the accession number or the marking is done using a permanent marker. ‘Embedding in Histopathology’
Advantages
- Since the cassette is processed with the tissues and afterwards used for embedding, the writing has to be done once
- Cassettes are thin so less wax is required.
- The space required for filing the blocks is less.
Disadvantages
- A special clamp has to be used in the microtome for this technique.
- The cassettes are shallow hence thin sections should be taken for processing.
PARAFFIN WAX ADDITIVES
Various substances can be added to paraffin wax in order to modify its consistency and melting point to improve the efficiency during microscopy. Additives increase the hardness of blocks. This helps in cutting thinner sections at higher temperature. Stickiness of the medium is increased so better ribbons can be obtained. However if larger quantities of additives are added, undesirable side effects may be seen. ‘Embedding in Histopathology’
Commonly used additives
Ceresin – It is hard white substance derived from mineral ozokerite. Its melting point is between 61 to70o C. The addition of 0.3-0.5% is sufficient to reduce the crystalline structure of paraffin wax. ‘Embedding in Histopathology’
Bees’ wax – It is yellow substance with melting point of 64o C. This also educes the crystalline structure of the paraffin wax and improves the ribbon quality.
Bayberry wax – It is a vegetable wax and present in the peal of bayberry. It is extracted from the peal of the fruit. Its melting point is 45° C.
Devices for tissue embedding
Devices designed specifically for tissue embedding are available for laboratories in need of such equipment. These machines vary in size and design depending on the number of samples they are designed to process. Some are designed for specific embedding media, including proprietary compounds intended for specific kinds of histopathology applications. Tissue embedding equipment tends to be expensive. Manufacturers have sales representatives who can provide information and advice when a lab is selecting new or replacement equipment. ‘Embedding in Histopathology’
Tissue embedding machine
All the blocking steps can be performed with the help of tissue embedding machine. The embedding machine contains the following parts –
- Mould warmer, cassette bath, working surface warmer with a nozzle for pouring the wax, forceps well and cold plate. ‘Embedding in Histopathology’
- The cold plate is of high efficiency refrigeration system having temperature control ranging from different freezing points to 4 or 5 degree C. It can occupy about 50-60 blocks.
- Large 3-5 litre capacity paraffin reservoir with adjustable temperature of 45-75 degree C.
- Optional vacuum lids, which allows for vacuum infiltration of tissues.
- It has a forceps warmer convenient drain for excess wax.
- The embedding machines are available with many other features.
Method of Embedding
- Open the tissue cassette, check requisition form entry to ensure the correct number of tissue pieces is present.
- Select the mould; there should be sufficient room for the tissue with allowance for at least a 2 mm surrounding margin of wax. Leuckhart mould method-This is the traditional embedding method. The “L moulds are adjusted according to the shape and size of the tissue. Glycerine may be applied to the L pieces and also to the metal or glass
plate on which the moulds are placed for embedding. Simple glossed wall
or floor tiles may also be used in place of glass plate. - Fill the mould with paraffin wax. ‘Embedding in Histopathology’
- Using warm forceps select the tissue, taking care that it does not cool in
the air; at the same time. ‘Embedding in Histopathology’ - Place the tissue in the mould according to the side to be sectioned. This
side should be facing down against the mould. A small amount of pressure
may be used in order to have more even embedding. - Chill the mould on the cold plate, orienting the tissue and firming it into
the wax with warmed forceps. This ensures that the correct orientation is
maintained and the tissue surface to be sectioned is kept flat. - Insert the identifying label or place the labelled embedding ring or cassette
base onto the mould. ‘Embedding in Histopathology’ - Add more paraffin into the mould to fill the cassette and mould.
- Cool the block on the cold plate.
- Remove the block from the mould. ‘Embedding in Histopathology’
- Cross check block, label and requisition form.
Orientation of different tissue Embedding in Histopathology
During embedding the orientation of tissue is important. Correct orientation of tissue in a mould is the most important step in embedding. Incorrect placement of tissues may result in diagnostically important tissue elements being missed or damaged during microtomy. During embedding it is important to orient the tissue in a way that will provide the best information to the pathologist. At the time of grossing, mark with India ink may be put on the side of the tissue opposite that to be cut. The embedding should be done according to the type of tissue. The requisition form should always be read during embedding for proper orientation.
