INFILTRATION

HISTOPATHOLOGY LECTURE
By: MA. THERESA FEDOC – MINGUITO, MD, DPSP-CP
STEPS IN
TISSUE
PROCESSING
  IMPREGNATION

 Process whereby the clearing agent is completely removed from

INFILTRATION the tissue and replaced by a medium that will completely fill all
the tissue cavities and give a firm consistency to the specimen

 allows easier handling and cutting of suitably thin sections without

any damage or distortion to the tissue and its cellular components
  soluble in processing fluids
 suitable for sectioning and ribboning
 molten between 30°C and 60°C
 translucent or transparent / colorless
 stable
CHARACTERISTICS OF AN
IDEAL INFILTRATING (and  homogeneous
embedding) MEDIUM
 capable of flattening after ribboning
 non-toxic
 odorless
 easy to handle
 inexpensive
TYPES OF
1. Paraffin wax
INFILTRATING 2. Celloidin (collodion)
(and 3. Gelatin
embedding) 4. Plastic
MEDIUM
PARAFFIN  simplest, most common
WAX  a polycrystalline mixture of solid hydrocarbons produced during
the refining of coal and mineral oils
 solid at room temperature but melts at temperatures up to about
65°C or 70°C
 most common melting point for histological use is 56°C to 58°C

 at its melting point, it tends to be slightly viscous, but this

decreases as the temperature is increased
 paraffin wax is used at about 2°C above its melting point
 increase the temperature to above 60°C or 65°C to decrease
viscosity and improve infiltration
  After being completely cleared, the tissue is submerged in two or
more changes of melted paraffin wax, either in a paraffin oven or
in an incubator which has been regulated at 55-60°C
Procedure for  duration and number of changes required for thorough
infiltration / impregnation of tissue depends on:
 Size and type of tissues: Longer time is required for thicker tissues.
impregnation  Use of vacuum imbedding: Vacuum reduces the time required for
complete impregnation.
 Clearing agent employed
ways by which
paraffin wax 1. manual
impregnation 2. Automatic

may be 3. vacuum

performed:
 Fixation:
10% Buffered Formalin 24 hours
Dehydration:
MANUAL 70% Alcohol 6 hours
PROCESSING 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
AUTOMATED
PROCESSING
VACUUM
EMBEDDING
  reduces the time when tissues are subjected to high temperatures
thus minimizing heat-induced tissue hardening
 facilitates complete removal of transition solvents
 prolongs the life of wax by reducing solvent contamination
 vacuum hastens the removal of air bubbles and clearing agent
VACUUM from the tissue block, thereby promoting a more rapid wax
penetration of the tissue
EMBEDDING  particularly recommended for urgent biopsies, for delicate tissues
such as lung, brain, connective tissues, decalcified bones, eyes,
spleen and central nervous system
 vacuum infiltration requires a vacuum infiltrator or embedding
oven, consisting of wax baths, fluid trap and vacuum gauge, to
which a vacuum of up to 760 mm Hg is applied using a water or
mechanical pump
  of the three methods of paraffin wax impregnation, vacuum
impregnation gives the fastest result.

PARAFFIN WAX  total impregnation time, however, generally depends upon the
nature and size of the tissues to be processed, and the type of
IMPREGNATION clearing agents to be used.
 larger and denser tissue blocks (e.g. bones, fibroids, brains) usually
require longer periods and more frequent changes of wax.
  prolonged treatment in melted paraffin causes shrinkage and
hardening of tissues, making cutting difficult, the tissue should
not be left in the paraffin oven for more than 4 hours
 the shorter the time in the hot oven with adequate paraffin
impregnation and evaporation of clearing agent, the better it is for
the tissue
Practical  tissues become increasingly harder and more brittle as they are
Considerations heated
 infiltration in overheated paraffin (above 60°C) 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°C 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°C higher than its melting point
 wax that has been trimmed away from the impregnated tissue
may be melted and filtered for future use, with a coarse filter
Practical paper, e.g. Green's No. 904
 when wax has been reused, some amount of water inevitably is
Considerations 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°C,
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
Practical  for fixed knife microtomes, a relatively hard wax with a higher
Considerations melting point is recommended
 heavier microtome knives require harder paraffin wax than lighter
ones
  PARAPLAST
SUBSTITUTE  EMBEDDOL
FOR  ESTER WAX
PARAFFIN  WATER SOLUBLE WAXES
 CARBOWAX – MOST COMMON
WAX  DIMETHYL SULFOXIDE (DMSO)
TYPES OF
1. Paraffin wax
INFILTRATING 2. Celloidin (collodion)
(and 3. Gelatin
embedding) 4. Plastic
MEDIUM
  Celloidin (Collodion) 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
CELLOIDIN cellulose dissolved in equal parts of ether and alcohol.
IMPREGNATION  This is used mainly for preparing soft tissue sections of mixed
consistency such as eyes and brain.
 Two methods:
 WET CELLOIDIN METHOD
 DRY CELLOIDIN METHOD
CELLOIDIN
IMPREGNATION
TYPES OF
1. Paraffin wax
INFILTRATING 2. Celloidin (collodion)
(and 3. Gelatin
embedding) 4. Plastic
MEDIUM
  rarely used except when dehydration is to be avoided and when
tissues are to be subjected to histochemical and enzyme studies
 used as an embedding medium for delicate specimens and frozen
tissue sections
 prevents fragmentation of tough and friable tissues when frozen
GELATIN sections are cut
IMPREGNATION  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
 has a low melting point and does not cause over-hardening of
tissues 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
GELATIN  and finally to another fresh solution of 20% gelatin with 1% phenol

IMPREGNATION allowed to cool in a refrigerator until impregnation and embedding
are completed

 The volume of the impregnating medium should be at least 25

times the volume of the tissue.
 - END -
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