RAPID PROCESSING TECHNIQUE
HISTOPATHOLOGY-LABORATORY
RAPID PROCESSING TECHNIQUE
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RAPID TISSUE PROCESSING
ADVANTAGES
1. Early diagnosis for patient
▪ Urgent cases:
✓ Transplant patients and others
benefit from prompt adjustments
on the treatment based on early
biopsy report.
2. Elimination of toxic chemicals from the
work environment, and elimination of
requirements to monitor the fumes in the
environment.
3. Immunohistochemical reactions are not
adversely affected, instead, enhanced
after rapid tissue processing
4. With appropriate fixatives, molecular
analyses for DNA, RNA, proteins can be
performed from paraffin blocks.
Done when there is an urgent need for an
immediate histopathologic evaluation
Done when some specimens and some
diagnosis for that matter are not amenable to
frozen section study
Applicable to small specimens only
Improvements:
o Microwave incorporate in manual,
semi-automated, and fully automated
systems
o Incorporating computer control of the
microwave “dosage”
o Temperature
o Vacuum paraffin impregnation
PRINCIPLE OF MICROWAVE
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Microwave excitation of the molecules
increases movement in both solutions and
tissues
improved tissue penetration and fixation and
preservation of tissue antigens of interest.
Size of tissue: up to 1.5 mm thick
DISADVANTAGES
1. Sections must be thin enough so fixative
and dehydrating solutions can penetrate
completely which increases time required
in grossing of specimens.
2. Certain types of tissues may require
additional steps before placing into the
rapid tissue processor.
3. Continuous flow processing eliminates
batching of specimens and necessitates
ongoing attention to the instrument, since
as samples complete the processing
cycle, they must be removed from the
instrument to accommodate the next
basket of cassettes.
4. Microwave ovens specifically designed
for tissue processing are nor preferred
EQUIPMENT
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Kitchen knife
Microwave oven
RAPID TISSUE PROCESSOR
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Enclosed system similar in configuration to
modern vacuum assisted conventional tissue
processor.
Reduces processing time to 3 hours when
combined with microwave fixation
Run in batch modes
Small blocks such as those from endoscopic
biopsies are processed in an abbreviated
protocol.
Large blocks require special procedure or
handling
MICROWAVE FIXATION
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CONTINOUS RAPID TISSUE PROCESSOR
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Allows for the addition of new specimens to
the process every 15 minutes as a reaction
chamber becomes available.
ADVANTAGES:
o Reagent volumes are small
o All reagents are inexpensive
o Reagents are non- toxic
Non-formaldehyde fixatives are better in this
fixation.
Alcohol based fixative when combined with
formalin-free microwave-based tissue
processing permits recovery of *DNA, RNA
and proteins for molecular analyses.
UNIVERSAL MOLECULAR FIXATIVE – UMFIX
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Mixture of methanol and polyethylene glycol
Tissues exposed to UMFIX have their
morphology comparable to formalin-fixed
tissues.
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FROZEN SECTIONS
ADVANTAGES:
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Improved antigen preservation in tissue
sections
Elimination of noxious formalin fumes from the
workplace.
MICROWAVE PROCESSING
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Based on the principle of using the
microwave energy to speed up the diffusion
of fluids into and out of the tissues.
Microwave heating stimulates alcohol
diffusion in histologic processing.
It also allow the use of alcohol during
dehydration using one step only
Paraffin must be added to the microwave in
liquid form.
o Melted with the intermedium at:
▪ 82 0C for ethyl alcohol
▪ 78 0C for isopropanol
Purpose: “Flash evaporation of the alcohol or
propanol”
o Alcohol quickly heats and dissipates
while the paraffin remains inert,
allowing paraffin to fully infiltrate the
specimen, eliminating the use of
xylene.
HISTOPATHOLOGY-LABORATORY
FROZEN SECTIONS
USES
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PREPARING TISSUE FOR FREEZING
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MICROWAVE STAINING
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Microwave reduced significantly the time
required for staining of tissue, particularly
when using heavy metals.
Equipped with temp probes and air-bubble
agitation device.
Better contrast
More intense staining
Compared with conventional metal staining
methods
Optimum temperature:
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Metallic stains - 75 0C to 95 0C
Non-metallic stains – 55 0C to 6
Tissue for freezing should be frozen or fixed as
promptly as possible after cessation of
circulation to avoid morphological distortions
and damage due to:
o Tissue drying
o Autolysis
o Self-digestion.
o Putrefaction
The object is to freeze so rapidly that water
does not have time to form crystals and
remains in a vitreous form that does not
expand when solidified.
METHODS FOR PREPARING FROZEN SECTION
1. Cold knife procedure
2. Cryostat procedure using cold microtome
Microwaved slides have:
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Rapid pathologic diagnosis during surgery,
Diagnostic and research enzyme
histochemistry
Diagnostic and research demonstration of
soluble substances,
Immunofluorescent and
immunocytochemicals staining,
Some specialized silver stain
COLD KNIFE PROCEDURE
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Tissue should be frozen and be firm enough
for sectioning.
The tissue is then lifted manually to the knife
and trimmed until the surface is flat.
Surface of the tissue is then warmed with the
finger until the frozen hard tissue starts to thaw
and becomes visible to the naked eye,
known as the Dew Line.
o The point at which tissue may then be
cut at 10 micra thickness
Sections do not form ribbons, instead stick to
the knife blade, and should be removed by a
camel brush or finger moistened with water.
Transfer sections to a dish of distilled water to
separate and be picked up individually for
mounting and staining.
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Use water dish in a dark or black background,
in order to see the sections which are usually
colorless or very light in color.
Tissues that are frozen too hard will usually
chip into fragments when cut.
Soften by warming slightly with the ball of the
finger or thumb.
Tissues not sufficiently frozen will cut thick and
crumble, and the block may come away
from the stage.
Certain temperature between the block and
knife is employed. The knife being colder.
o Knife ------------- 40° to -60° C
o Tissue ------------ 5° to – 10° C
o Environment ----0° to – 10° C
Success of the procedure depends upon:
o Ambient temperature
o Humidity
ISOPENTANE
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Isopentane is liquid at room temperature
AEROSOL SPRAYS
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Adequate for freezing small pieces of tissue
except muscle
NOTE: Success of fresh tissue sectioning depends on
the temperature both the tissue and the knife.
MOUNTING OF TISSUE BLOCK IN CRYOSTAT
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Mounting media: Synthetic water-soluble
glycols and resins
EXAMPLE: OCT compound
CRYOSTAT PROCEDURE
EQUIPMENT USED: CRYOSTAT
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A chamber consisting of an insulated
microtome housed in an electrically driven
chamber maintained at a temperature near 20°C.
Optimum working temp: - 18° to – 20°C
Majority of sections can be cut in an
isothermic conditions
MORE COMMONLY USED METHODS OF FREEZING
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Liquid nitrogen
Isopentane cooled by liquid nitrogen
Carbon dioxide gas
Aerosol sprays
LIQUID NITROGEN
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Used in histochemistry
Intraoperative procedures
Most rapid of commonly available freezing
agents.
DISADVANTAGE:
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Soft tissue is liable to crack due to the rapid
expansion of the ice within the tissue.
It also overcools urgent biopsy blocks,
Tissue snap-frozen in liquid nitrogen must be
allowed to equilibrate to cryostat chamber
temperature before sectioning is attempted.
Remedy: freeze the tissue in isopentane, O.C.T.
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Thickness: 2 -4 mm to minimize knife hitting
the metal tissue block holder
Small tissues such as curettings or brain
biopsies– place in of thick base OCT cpd.
Then surrounded by additional OCT cpd and
frozen by liquid nitrogen.
Both the tissue block and the microtome knife
are left in the cryostat for 15 to 30 minutes at 20C, to ensure that they are cooled to
correct temperature
Sections 5 – 10 micra are cut slowly and
steadily,
Remove from the knife using a camel
hairbrush,
Attached to slides of cover-glasses at rm
temp.,
Air Dry,
Fix,
Stain,
Mounting cryostat sections.
CARE OF MICROTOME
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The cryostat should be always left on.
Time for the knife to come down to operating
temperature - 1 hour
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Spare knife should always be kept inside the
cryostat cabinet.
To ensure that the sections will cut smoothly
and freely unto the knife surface, the knife,
the undersurface, anti-roll plate must be kept
clean and dry.
Clean knife and anti-roll plate
DISADVANTAGE
1. This method is time-consuming and
expensive:
o Drying is the most consuming part of
the process.
2. Freeze-dried materials are diff to section
than ordinary paraffin blocks. Brittle and
inadequately supported due to short
period wax impregnation. Water must be
avoided.
o Preference: warm alcohol, acetone
and or mercury
SPECIAL PROCESSING TECHNIQUES
METHODS USED IF CHEMICAL FIXATION IS TO BE
AVOIDED
1. Freeze drying
2. Freeze substitution
COMMON PRINCIPLE
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Rapid preservation of the tissue block by
freezing to produce instant cessation of
cellular activity thereby preventing chemical
alteration of tissue constituents and
displacement of cellular tissue components.
Freezing agents: Liquid nitrogen
Agents that give higher conductivity
o Isopentane
o Propane
o Pentane
o Dichloro-difluoromethane
OUTSTANDING GOOD FEATURES
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USES
1. Demonstration of hydrolytic enzymes,
mucus substances, glycogen and
proteins.
2. Immunocytochemistry
3. Fluorescent antibody studies of
polypeptide and polypeptide hormones
4. Autoradiography
5. Formaldehyde-induce fluorescence of
biogenic amines .
6. Scanning electron microscopy
FREEZE DRYING
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Rapid freezing at Temp: -160° C
Sub followed by desiccation by a physical
process of transferring the still frozen tissue
block in a vacuum at higher temperature
without the use of any chemical fixative.
Restricted to research laboratories
METHOD:
o 1- 2mm thick tissue – immersed in
isopentane or propane-isopentane
mixture
o Chilled to -160° C to – 180° C with
liquid nitrogen
o Solidification occurs in 2 – 3 sec
Transfer frozen section to a high vacuum
drying apparatus maintained at a temp. of 30° to -40° C depending upon the size of the
tissue.
Desiccation by: Sublimation, dehydration of
the tissue within 24 – 48 hours.
Once drying is completed, tissue is removed,
fixed, embedded.
Sectioning of tissue in routine manner
Staining
Minimal chemical change on the cells, mostly
esp. on the protein components
Less displacement of tissue and cell
constituents.
Avoids destruction of enzymes
FREEZE-SUBSTITUTION
Same with freeze drying except that:
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Instead of dehydrated in an expensive
vacuum drying apparatus, it is fixed in
Rossman’s formula or in 1% Acetone and
dehydrated in absolute alcohol.
Infiltration and embedding is the same as
freeze-drying.
Alternative form
Involves rapid freezing of tissue to – 160° C in
isopentane supercooled in liquid nitrogen.
Cryostat sections are cut 8 – 10 μm
Transfer to water-free acetone and cooled to
-70°C for 12 hours.
Sections are floated on to the coverslips or
slide
ADVANTAGE
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More economic
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Less time-consuming than freeze-drying
OVERALL ADVANTAGE OF CRYOSTAT SECTIONS:
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Simplest
Quickest
Least labor-intensive for producing frozen
sections
Intraoperative and rapid diagnosis of surgical
specimens.
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