Impregnation / Infiltration, & Embedding and Trimming of Tissue Block- MIDTERMS L10

IMPREGNATION

• Also known as INFILTRATION

• Process whereby the clearing agent is completely removed from the tissue & replaced by a medium that will completely fill all the tissue cavities, thereby giving a firm consistency to the specimen

• Process of replacing the clearing agent with the infiltrating medium.

• The medium used to infiltrate the tissue is usually the same medium used for embedding

FOUR TYPES OF IMPREGNATION AND EMBEDDING MEDIA

1. Paraffin wax Impregnation

2. Celloidin (Collodion) Impregnation

3. Gelatin Impregnation

4. Plastic

BUTSCHLII

The man who introduced paraffin wax embedding

PARAFFIN

• Simplest, most common and the BEST infiltrating/ embedding medium.

• Process is very rapid, allowing sections to be prepared within 24 hrs

PARAFFIN DISADVANTAGE

• Overheated paraffin makes the specimen brittle

• Prolonging will cause excessive tissue shrinkage and hardening

• NOT recommended for fatty tissues (the dehydrants and clearing agents used in the process dissolve and remove fat from the tissues).

AFTER CLEARING

Tissue is submerged in 2 or more changes of melted paraffin wax.

TEMPERATURE OR PARAFFIN OVEN

55-60*C

(Paraffin oven must be maintained at a temperature 2-5*C above the MP of the paraffin wax to be used.)

MELTING POINT OF COMMON WAXES

45, 52, 56, 58°C

WAX WITH MELTING POINT OF 56*C

Is normally used for routine work.

LAB TEMPERATURE OF 20-24*C

Paraffin wax melting point is 54-58*C

LAB TEMPERATURE OF 15-18*C

Melting point of wax 50*C to 54*C

HARD TISSUE

Require wax with higher melting point

WHEN WAX HAS BEEN REUSED

Some water is mixed with it

IF EXCESSIVE WATER ACCUMULATES

This may impair the impregnating capacity of the medium.

TO REMOVE EXCESS WATER

Heat the wax to 100-105*C

• (Paraffin wax may be used twice only!)

THREE WAYS BY WHICH PARAFFIN WAX IMPREGNATION AND EMBEDDING MAY BE PERFORMED

• Manual Processing

• Automatic Processing

• Vacuum embedding

MANUAL PROCESSING

AUTOMATIC PROCESSING

With the use of autotechnicon

AUTOTECHNICON

• Fixes, dehydrates, clears and infiltrates tissues

• For rapid diagnosis with less technicality

• 2-3 changes of wax is required

VACUUM EMBEDDING

• Involves the wax impregnation under negative atmospheric pressure (400-500 mmHg) inside an embedding oven.

• Gives the fastest results

• To hasten removal of air bubbles and clearing agent

• Promote rapid wax penetration

• Time reduce from 25-75% of the normal time require

FACTORS AFFECTING PARAFFIN WAX IMPREGNATION

• Nature and size of the tissues to be processed

• Type of clearing agents to be used

PRECAUTIONS OBSERVED IN PARAFFIN WAX IMPREGNATION

• Tissue should not be left for longer periods of time

• Maintained a temperature 2 to 5°C above the melting point

• Paraffin wax must be pure

• Fresh wax should be filtered before use

• When using automatic tissue processing machine, wax usually becomes admixed with the clearing agent, especially in the first beaker

• For fixed knife microtomes, a relatively hard wax with a higher melting point is recommended

SUBSTITUTES FOR PARAFFIN WAX

• Paraplast

• Ester Wax

• Water Soluble waxes

PARAPLAST

Mixture of highly purified paraffin and synthetic plastic polymers

MELTING POINT OF PARAPLAST

56-57*C

TYPES OF PARAPLAST

• Embeddol

• Bioloid

• Tissue mat

EMBEDDOL

Melting point 56-58°C

BIOLOID

Semisynthetic recommended for embedding eye

TISSUE MAT

Product of paraffin

ESTER WAX

• Has lower melting point 46-48°C

• Harder than paraffin

• Not soluble in water but soluble in 95% Ethyl Alcohol

WATER SOLUBLE WAXES

• Mostly polyethylene glycols

• MELTING POINT: 38-42°C or 45-56°C

CARBOWAX

• Most common water-soluble wax (hydroscopic)

  • 4 changes of carbowax for routine processing

  • 70%,90%,100% (2x)

  • 56°C at 30 minutes

  • 45 minutes and 1 hour (with agitation)

CELLOIDIN (COLLODION) [CELLOIDIN IMPREGNATION]

• Purified form of nitrocellulose soluble in many solvents

• Suitable for specimen with hollow cavities

• Recommended for processing neurological tissues

• Avoiding the crumbling of tissues during sectioning

• Does not require heat

DISADVANTAGE OF CELLOIDIN IMPREGNATION

• Very slow (lasting for several days or weeks)

• Very thin section difficult to cut

• Photomicrographs are difficult to obtain

• Very volatile

TWO METHODS FOR CELLOIDIN IMPREGNATION

1. Wet Celloidin

2. Dry Celloidin

WET CELLOIDIN

Recommended for bones,teeth, large brain sections and whole organs.

DRY CELLOIDIN

Preferred for processing of whole eye sections

NITROCELLULOSE METHOD [CELLOIDIN IMPREGNATION]

Low Viscosity Nitrocellulose (L.V.N.)

LOW VISCOCITY NITROCELLULOSE (L.V.N.)

• Is another form of celloidin soluble in equal concentration of ether and alcohol, with lower viscosity, allowing it to be used in higher concentration and still penetrate tissue rapidly

• It forms a harder tissue block and makes cutting of thinner sections possible

• The tendency to tissues to crack may be prevented by adding plasticizers (oleum ricini or castor oil) when embedding chrome-mordanted tissues.

• L.V.N. is more explosive than celloidin.

GELATIN IMPREGNATION

• Rarely used except when dehydration is to be avoided and when tissue are subjected to histochemical and enzyme studies

• Used as an embedding medium for delicate specimens and frozen tissue sections.

• It is water soluble, does not require dehydration and clearing, although fixatives should still washed out by running water.

• Tissue is placed in 10% gelatin with 1% phenol for 24 hours, transferred to 20% gelatin with 1% phenol for the next 12 hours, 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.

• Tissue should not be more than 23 mm. thick

• The volume of the impregnating medium should be at least 25x the volume of the tissues.

PLASTIC (RESIN)

• It has provided superior results for light microscopic studies, particularly in hard tissues

• Tissue sections must be 4-6um, such in renal biopsies and bone marrow biopsies.

• Plastic are classified into epoxy, polyester, or acrylic, based on their chemical composition

EMBEDDING

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.

ORIENTATION

Process by which the tissue is arrange in precise position in the mold during embedding, on the microtome before cutting and on the slide before staining

OTHER EMBEDDING METHODS

1. Celloidin or Nitrocellulose method

2. Double Embedding Method

CELLOIDIN OR NITROCELLULOSE METHOD

Recommended for embedding hard tissues

DOUBLE EMBEDDING METHOD

• Process in which tissues are first infiltrated with celloidin and subsequently embedded in paraffin mass.

• Used to facilitate cutting of large block of dense firm tissue like the brain.

TYPES OF BLOCKING- OUT EMBEDDING MOLDS

1. Leuckhart’s embedding mold

2. Compound embedding Unit

3. Plastic embedding rings & base molds

4. Disposable embedding molds

   • Peel-away

   • Plastic Ice trays

   • Paper boats

LEUCKHART’S EMBEDDING MOLD

Consist of two L-shaped strips of heavy brass of metal arranged on a flat metal plate and which can be moved to adjust the size of the mold to the size of the specimen

COMPOUND EMBEDDING UNIT

Made up of a series of interlocking plates resting on a flat metal base, forming several compartments

PLASTIC EMBEDDING RINGS AND BASE MOLDS

Consist of a special stainless steel base mold fitted with a plastic embedding ring, which later serves as the block holder during cutting.

TRIMMING OF SECTIONS

Is the process of removing the excess wax by cutting off from the block to expose the tissue surface in preparation for sectioning.

ONCE THE WAX HAS SOLIDIFIED

• The wax block is removed from the mold

• The identification number is noted & the excess wax is cut off from the block to expose the tissue surface in preparation for cutting