HISTOPATHOLOGY

IMPREGNATION

Impregnation (Infiltration) – process of completely removing the clearing agents from the tissue and
replaced by a MEDIUM that will completely fill the tissue cavities giving the tissue:
1. Firm consistency
2. Easier handling of tissue and suitable cutting into thin sections without damage or
distortion

GENERAL TYPES
A. Paraffin Wax Impregnation
 Simplest, most common, routinely used
 Best embedding medium used for routine tissue processing
 Melting point : 56°C – used for routine work
 Common wax melting pt. = 45°C, 52°C, 56°C, 58°C
Advantages:
1. Compatible with many stains
2. Possible cutting/production of serial sections
3. Rapid process – within 2 hours
4. Indefinite storage of tissue block is possible without considerable tissue destruction
Disadvantages:
1. Overheated specimen – Brittle tissue
2. Prolonged impregnation – Shrinkage, hardening, difficult to cut tissue
3. Inadequate impregnation – Retention of clearing agents (soft, crumbled and broken spx.)
4. Long immersion for bones, teeth, brain, eyes
5. Paraffin processing is not recommended for fatty tissues

METHODS:

 By Manual Processing
 Oven temp. should be higher than 56°C, that is 55-60°C or 2-5 degrees higher
1. Four (4) changes of wax with 15minutes interval insure complete removal of clearing
agents from the tissue.
2. Immersed in another melted paraffin for 3 hours to insure complete embedding or
casting

 Automatic Processing
 Use of AUTOTECHNICON (e.g. Elliot Bench-Type Processor)
 Requires 2-3 changes only 1st -2nd – Formalin
 Results are consistent due to constant tissue agitation 3rd-5th – Ethanol
 12 individual processing steps 6th – 8th- Xylene
 Parts: 9th – 10th – Paraffin wax
 Ten (10) 1-liter glass beakers – where rgts. are placed
 Two (2) thermostatically controlled wax bath – protection against
OVERHEATING
 Transfer arm – transfers tissue to different processing rgt.
 Spring-loaded plunger – facilitates removal of cover plate
 Cover plate – Cover the beakers
 Electrical clock / Timing disc– controls the time needed for each processing step
- Sets transfer arm and mechanism into motion
 Types of Autotechnicon
 Tissue transfer / Dip and dunk
o Specimen-containing cassettes to be processed are moved from container to
container
o Vapors are not vented and directly inhaled by the histotechnologist
 HISTOPATHOLOGY

 Fluid transfer / enclosed type

o Cassettes stay in single process chamber or retort and fluid are pumped in & out as
required
o Specimen do not dry out and vapors are vented through filter
Precautions:
 Presence of odor in the clearing agent during final paraffin wax bath = paraffin wax should be
changed
 First 100% ethanol bath = DISCARDED
 Clearing agents and dilute ethanol = changed at least once a week
 Wax bath thermostat = at least 3 degrees above the wax melting pt.

 Vacuum Embedding
 Wax impregnation under negative atmospheric pressure inside an embedding oven
 Purpose: To hasten removal of air bubbles and clearing agents to promote rapid
penetration
 Recommended for: lung, brain, connective tissues, decalcified bones, eyes, spleen,
CNS
 Time required for complete impregnation = reduced from 25-75% of the normal tissue
processing
 Parts:
 Heavy brass chamber with glass slid = produces airtight seal
 Water-jacket = maintained at 2-4°C above the wax melting pt.
 Screw valves:
o 1st : allows readmission of air when the bath is under negative pressure
o 2nd: connected to a tube and allows exhaustion of 400-500mmHg
NOTE: degree of vacuum should not exceed 500mmHg
 Stopcock = prevent water from being sucked back
Procedure:
1. Clear in two changes of Xylene – 1hour each
2. Place tissue in a molten wax, vacuum chamber and airtight oven
3. Exhaust air slowly using vacuum pump or Venture suction pump until negative pressure of 400-
500 mmHg is obtained
4. Leave for 15 minutes then slowly readmit air until normal atm. pressure is reached
5. Place tissue in fresh wax
6. Repeat steps 3 & 4
7. Place tissue in fresh wax
8. Repeat step 3 and leave for 30-45 minutes
9. Bring to normal atm. Pressure and embed the tissue

FACTORS AFFECTING PARAFFIN WAX IMPREGNATION

 Vacuum impregnation – gives the fastest result
 Total impregnation time depends upon: Nature and size of the spx / Clearing agent to be used
 Benzene and Xylene – easily removed from tissues
 Chloroform and Cedarwood Oil – Difficult to remove and require frequent wax changes
 Addition of Benzene = hasten displacement of Cedarwood Oil with less tissue shrinkage
Precaution Observed in Paraffin Wax Impregnation
 Infiltration in overheated paraffin (above 60°C) = Shrinkage and hardening of tissue and complete
destruction of lymphoid tissues
Remember: To avoid this, maintain the paraffin oven temp. 2-5°C above the melting pt. of the paraffin
 Paraffin wax MUST be pure
 Fresh wax should be filtered before use in a wax oven at a temp. 2°C higher than its melting pt.
 Trimmed wax = filter using coarse filter paper (e.g Green’s No. 904)
 Reused wax = Remove water by heating the wax to 100-105°C
 HISTOPATHOLOGY

SUBSTITUTES FOR PARAFFIN WAX

1. Paraplast
 Mixture of paraffin and synthetic plastic polymers
 Melting point : 56-57°C
Advantages:
 Permits easier cutting of large dense tissue blocks like bones and brain
 Better ribboning of sections
 Does not tend to crack
 More uniform blocks are formed

2. Embeddol
 Similar to Paraplast
 Melting point : 56-58°C
Advantages:
 Less brittle and compressible than paraplast

3. Bioloid
 Semisynthetic wax recommended for embedding eye specimen

4. Tissue mat
 Product of paraffin, with rubber and with the same property as Paraplast

5. Ester Wax
 Harder than paraffin
 Melting point : 46-48°C
Advantages:
 Requires 3-4 changes of wax
 Done in a heavy-duty microtome (e.g Sliding or Sledge type microtome)
 Soluble in 95% ethyl alcohol but not in water
*Eliminate clearing but not dehydration
 Cellosolve or Xylene can be used as clearing agents

6. Carbowax / Polyethylene Glycol

 Melting point : 38-42°C or 45-56°C
 Four changes are made:
 70% - 30 minutes
 90% - 45 minutes @ 56°C, embedded in fresh Caarbowax at 50°C
 100% (2) – 1 hour
Advantages:
 Water soluble
 Eliminate dehydration and clearing
 Suitable for enzyme histochemical studies and cytological preservation
Disadvantages:
 Hygroscopic
 Difficult to float out and mount
- Purpose of float out bath: To remove wrinkles in the ribbon
*Remedy: Addition of soap to water or use of 10% Polyethylene Glycol 900 in water
 HISTOPATHOLOGY

B. Celloidin (Colloidion) Impregnation

 Nitrocellulose dissolved in ether and alcohol
 Suitable for specimens with large hollow cavities that tend to collapse
 Hard and dense tissues : bones and teeth
 Large tissue section : Embryo
Advantages:
 For processing of neurological tissues
 With rubber consistency that prevents distortion of tissue block
 Suitable for eye spx (retina detached from sclera and choroid)
Disadvantages:
 Very slow acting (days-weeks)
 Vapor of ether solvent is very flammable (*Reason why NO HEAT is applied when in use)
 Serial sections, thin sections, photomicrograph are difficult to produce

Methods:
a. Wet Celloidin Method
 Recommended for bones, teeth, large brain sections and whole organs

b. Dry Celloidin Method

 For eye spx
 Similar with WCM in terms of principle and procedure
 70% alc. is not used, instead Gilson’s mixture* is added before hardening to make tissues
transparent
*Gilson’s mixture = chloroform & cedarwood oil
Procedures (WET & DRY):
1. Ether and alcohol for 12-24 hours
2. Thin celloidin (2-4%) for 5-7 days
3. Medium celloidin (4-6%) for 5-7 days
4. Thick celloidin (8-12%) for 3-5 days
5. Removed from thick celloidin and transferred to an embedding medium with freshly poured thick
celloidin and kept in a jar or dessicator to evaporate the alc.-ether solvent
6. FOR WET: Tissue block is stored in 70-80% alcohol
FOR DRY: Tissue block is stored in a Gilson’s mixture

NITROCELLULOSE METHOD USING LVN

 Low Viscosity Nitrocellulose (L.V.N) – marketed while wet with alcohol
Advantages
 Soluble in equal conc. of ether and alcohol, Penetrates rapidly, Harder tissue block and possible
thin sections cutting
 Tendency to crack may be prevented by adding PLASTICIZERS (e.g oleum ricini or castor oil)
Disadvantages
 More explosive than celloidin. exposure to air and sunlight makes the alcohol to evaporate
 Destroyed when no longer used because it becomes dangerous deu to the evaporation of alcohol
 HISTOPATHOLOGY

C. Gelatin Impregnation
 For histochemical and enzymes studies
 For delicate specimens and frozen section
Procedure:
1. Wash out 10% formalin
2. Place tissue in 10% gelatin with 1% phenol for 24 hours
3. 20% gelatin with 1% phenol for 12 hours
4. Fresh solution of 20% gelatin with 1% phenol
5. 10% formalin for 12-24 hours for the hardening of tissues
*1% phenol prevents growth of molds

PLASTIC RESINS
 For light microscopic studies
 For hard tissues (undecalcified bones) and thin biopsies (renal biopsies and bone marrow)

Classifications:
1. Epoxy
 Mixture of epoxy plastics, catalysts, accelerators
a. Bisphenol A (Araldite) - Slow
b. Glycerol (Epon) – Low viscosity, mixture of isomers
c. Cyclohexane dioxide (Spurr) – obtained pure with very low viscosity and infiltrate fast
Disadvantages:
 Hydrophobic
 Composed mainly of Vinylcyclohexane dioxide (VCD), which is known to be carcinogenic
 Can produce sensitization through skin contact or inhalation

2. Polyester
 For electron microscopy

3. Acrylic
 For light microscopy
 Benzoyl peroxide – Catalyst that decomposes to form free phenol radicals – active site
for acrylic polymerization
a. Polyglycol methacrylate (GMA)
 Extremely hydrophilic
 Suitable for many stains
b. Methyl methacrylate (MMA)
 For undecalcified bone and other hard tissues
 HISTOPATHOLOGY

EMBEDDING

Embedding (Casting or Blocking) – process of placing the impregnated tissue into a MOLD contain a
medium allowing it to solidify.
 Embedding Medium – medium used in infiltration and embedding
 Orientation – process of arranging tissues in the mold, microtome, slide
Process:
1. Arranged at the bottom of the mold
2. Immersed in a melted paraffin at 5-10°C above its melting pt
3. Cooled rapidly in a refrigerator at -5°C or immersion in a cold water

REQUIREMENTS FOR EMBEDDING

A. EMBEDDING MOLDS
 Leukhart’s Embedding mold / Dimmock’s embedding irons or metal containers
 Consists of two L-shaped strips of heavy brass or metal
 Produce even, with parallel sides blocks
 Recommended for routine use but too slow or cumbersome in a busy laboratory

 Compound Embedding Unit

 Series of interlocking plates with several compartments

 Plastic Embedding Rings and Base Mold

 Special stainless steel base mold with plastic embedding ring* (*block holder during
cutting)
a. Tissue tek
 Warm plate – manage the impregnated spx
 Cold plate (-5°C) – for rapid solidification of the block
Advantages:
 Ease of use
 Less paraffin wax needed
 Faster embedding
 Permanent identification
 Produce easier orientation

 Disposable Embedding Molds

a. Peel away
 Molds are simply peeled off one at a time giving perfect even block without
trimming
b. Plastic Ice Trays
 Used in ordinary refrigerators, recommended for busy routine laboratories
 Removal of tissue block from the compartment by;
- Bending the plastic tray once wax has solidified
- Smearing the inner mold with glycerin or liquid paraffin
c. Paper Boats
 For embedding celloidin blocks
Advantages:
 Cheap and easy to make
 Provide easy and accurate identification of spx
 Possible rapid embedding of large or small vol. of spx
 HISTOPATHOLOGY

ORIENTATION
 Process of placing infiltrated tissue in a precise position (middle) in the mold
 Most important step in embedding
 Tissue are blocked with the surface to be cut facing down in a mold

Points to Consider in Orienting Tissue in the Block

1. Elongated tissue are placed diagonally across the block.
2. Tubular and walled spxs. as cysts, fallopian tubes and GI tissues are embedded en face so as to
provide cross sections showing all tissue layers.
3. Tissues with an epithelial surface such as skin are embedded to provide sections in a plane at a
right angle to the surfaces
4. Multiple tissue pieces are aligned across the long axis and the center of the mold, and not placed
randomly.

DOUBLE EMBEDDING – infiltrate tissue with celloidin and embed in paraffin

 Agar-paraffin embedding
 Tissues require external support or particular pre-embedment orientation
 Paraffin Wax Double Infiltration Methods
 Provide hard tissue additional support afforded by substances such as agar or
nitrocellulose with the convenience and ese of wax microtomy

TRIMMING

 Process of removing excess paraffin wax to form a four-sided prism